Protein-interaction network analysis reveals the role of Prp19 splicing factor in transcription of both intron-containing and intron-lacking genes
Katherine Dwyer, Mary-Ann Essak, Ahlam Awada, Zuzer Dhoondia, Athar Ansari
TL;DR
This study shows that the splicing factor Prp19 also plays a role in RNA polymerase II transcription, affecting both initiation and termination steps.
Contribution
The paper reveals a novel, splicing-independent role of Prp19 in the transcription cycle of RNAPII in budding yeast.
Findings
Prp19 interacts with TFIID, CPF, and RSC complexes in the chromatin context.
Depletion of Prp19 reduces transcription of both intron-containing and intron-lacking genes.
Prp19 affects preinitiation complex assembly and transcription termination.
Abstract
The process of transcription and cotranscriptional mRNA processing are facilitated by myriads of molecular interactions. To elucidate the protein-protein interactions that occur during transcription cycle of RNAPII, we performed mass spectrometry of affinity purified termination complexes from chromatin fraction. Quantitative proteomic analysis revealed interaction of termination factors with TFIIB, TFIID and SAGA complex. Furthermore, all three termination complexes displayed statistically significant interactions with Prp19, Prp43, Sub2, Snu114, Brr2 and Smb1 splicing factors. Since Prp19 consistently emerged as the interactor of both initiation and termination complexes, we affinity-purified the factor and performed mass spectrometry. Prp19 exhibited interactions with subunits of TFIID, CPF complex, and the RSC chromatin remodeling complex. These interactions were observed…
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Fig 7- —http://dx.doi.org/10.13039/100000057National Institute of General Medical Sciences
- —http://dx.doi.org/10.13039/100006710Wayne State University
- —http://dx.doi.org/10.13039/100006710Wayne State University
- —http://dx.doi.org/10.13039/100006710Wayne State University
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Taxonomy
TopicsRNA Research and Splicing · Genomics and Chromatin Dynamics · Fungal and yeast genetics research
Introduction
In the complex landscape of eukaryotic gene expression, the process of transcription by RNA polymerase II is intricately linked to the processing of nascent mRNA by capping, splicing, and cleavage-polyadenylation [1,2]. The processing of mRNA predominantly occur cotranscriptionally. The different steps of transcription and cotranscriptional RNA processing occur in a coordinated fashion and are fine-tuned by a complex network of molecular interactions. The initiation and termination steps have been shown to influence each other due to extensive promoter-terminator crosstalk often facilitated by the direct interaction of promoter-and terminator-bound factors [3–18]. The general transcription factor (GTF) TFIIB, for example, exhibits interaction with termination factors both in yeast and humans [19–21]. A complex comprising the TFIID and 3’ end processing-termination factors has been purified from mammalian cells as well [22]. Furthermore, Mediator complex subunits have been shown to interact with termination factors and affect termination of transcription in yeast and humans [18,23]. The promoter-terminator crosstalk often results in the formation of a looped gene architecture [3–5,7,8,13,18,20,23,24].
Recently, our laboratory conducted mass spectrometry analyses of affinity-purified initiation and termination factor complexes from chromatin fractions, to explore the protein-protein interactions that occur during transcription. These investigations revealed novel associations of TFIIB and Rat1 with splicing factors [21,25]. The interaction of transcription factors with splicing factors suggests a broader, yet currently unidentified, role of these molecular players in transcription and cotranscriptional mRNA processing. Given the cotranscriptional nature of splicing, it is reasonable to expect the interaction of splicing factors with components of the transcription machinery. The idea is corroborated by known functional interactions between splicing factors and transcription machinery in higher eukaryotes [26–31]. Since over 90% of protein-coding genes contain introns in mammalian systems, the interaction of spliceosomes with transcription machinery is not surprising. In contrast, less than 4% of yeast genes contain introns [32]. Despite that, transcription factors still exhibit genetic interaction with splicing factors in budding yeast [33–34]. It is tempting to speculate that splicing factor-transcription factor interaction plays a broader, possibly splicing-independent role in transcriptional regulation in eukaryotes. One of the splicing factors that we consistently detected in our proteomic analyses of GTFs was Prp19. Prp19 is a part of NineTeen complex (NTC) [35,36], which is an evolutionarily conserved complex with homologs reported in both yeast and humans. It facilitates catalytic activation of the spliceosome [37–41]. The Prp19 complex also interacts with the THO complex in yeast, which is a subcomplex of TREX (TRanscription-EXport), a conserved complex that couples transcription to nuclear mRNA export [42–44].
To explore the broader role of Prp19 in transcription and cotranscriptional RNA processing, we performed proteomic analysis of Prp19. Our results revealed multiple interactions of Prp19 with components of the RNAPII transcription machinery. The interactions were statistically significant and implicated Prp19 in transcription of protein-coding genes. Auxin-mediated depletion of Prp19 revealed its moderate stimulatory influence on the transcription of a subset of both intronic and non-intronic genes. Specifically, Prp19 crosslinked to the promoter, coding and terminator regions of genes and facilitated multiple steps of RNAPII transcription cycle. These findings challenge the traditional view of splicing factors solely as mediators of RNA processing, suggesting an additional possibly splicing-independent function in fine tuning of transcription by RNA polymerase II.
Results
Our previous work revealed that the interaction of TFIIB with termination factors is crucial for promoter-terminator communication through gene looping and plays a pivotal role in transcription termination [45]. Interestingly, TFIIB and the Rat1 termination factor also exhibit interactions with several splicing factors in the chromatin context [21,25]. To further explore the protein-protein interactions that take place during transcription, we affinity purified all three termination complexes of yeast: CPF, CF1, and Rat1, from soluble and chromatin fractions of yeast cell lysate (Fig 1A). Specifically, CPF was purified from a strain with Myc-tagged Ssu72, CF1 from a strain with HA-tagged Rna15, and Rat1 from a strain with HA-tagged Rat1. Mass spectrometry was performed to identify interacting protein partners of all three complexes. The results revealed some expected interactions of termination factors with RNAPII and the GTFs, and some rather novel interactions with splicing factors.
Prp19 interacts with RNAPII, general transcription factor TFIID, RSC chromatin modifiers and components of the TREX complex in chromatin fraction.(A) The workflow for purification of Prp19 from chromatin and soluble fractions. (B) Prp19 exhibits strong interaction with nine of the twelve subunits of RNAPII in the chromatin fraction. (C) Prp19 associates with four subunits of the general transcription factor TFIID. (D) Prp19 interacts with multiple subunits of the CPF termination complex. (E) Prp19 interacts with almost entire RSC chromatin remodeling complex. (F) Prp19 interacts with two subunits of the TREX complex. p-values calculated by two tailed t-test indicate significant enrichment of the different factors in chromatin fraction relative to soluble fraction. One asterisk () signifies a p-value equal to or smaller than 0.05 (p ≤ 0.05); two asterisks () signify a p-value equal to or smaller than 0.01 (p ≤ 0.01); while three asterisks () signify a p-value equal to or smaller than 0.01 (p ≤ 0.001). Error bars represent one unit of standard deviation based on four biological replicates. The method for calculating NSAF is described in detail in ‘Proteomic Analysis’ section of ‘Materials and Methods.
CPF, CFI and Rat1 termination complexes interact with RNAPII and the general transcription factors
It is known that all three termination complexes are recruited to chromatin cotranscriptionally as RNAPII reaches the 3’ end of a gene. We, therefore, assessed the presence of RNAPII subunits in the affinity purified termination complex preparations. All three termination complexes exhibited interaction with multiple RNAPII subunits (Fig A in S1 File). Notably, these interactions were observed exclusively in the chromatin derived preparations of termination factors (Fig A in S1 File, blue bars). We next investigated interactions of termination complexes with the general transcription factors. The results corroborated the previously published interaction of CF1 and Rat1 complexes with TFIIB (Fig B, panels B and C in S1 File). Additionally, all three termination complexes interact with subunits of the TFIID complex (Fig B, panels A, B and C in S1 File). Combined these findings presented evidence of a novel role of TFIID in promoter-terminator crosstalk, with TFIID engaging in stronger interactions with all three termination complexes compared to TFIIB. These results corroborated the preliminary evidence from earlier studies that there is extensive crosstalk between promoter and terminator-bound factors during transcription.
Termination complexes also interact with splicing factors
In yeast, splicing often occurs rapidly within seconds following the transcription of an intron, while the polymerase is just a few nucleotides downstream of the 3′ splice site [46–48]. Given the cotranscriptional nature of splicing, we reasoned that factors involved in initiation, elongation, and termination of transcription may physically or functionally interact with splicing factors. Consistent with this idea, we recently demonstrated interactions between the general transcription factor TFIIB and the termination factor Rat1 with several splicing factors in the affinity-purified preparations [21,25]. We therefore investigated the presence of splicing factors in our purified termination factor preparations from both chromatin and soluble cellular fractions. The results revealed that all three 3’ end processing-termination complexes interact with splicing factors (Fig C, panels A, B and C in S1 File). Most of these interactions were observed exclusively in the chromatin context (Fig C, panels A, B and C in S1 File, dark blue bars). Notably, the NSAF (Normalized Spectral Abundance Factor) values for these interactions ranged from 0.05 to 0.2, suggesting that splicing factors were present in sub-stoichiometric amounts in the termination factor preparations. It is particularly notable that all three termination complexes interacted with a similar set of splicing factors, including Prp19, Prp43, Snu114, Brr2, and Smb1 (Fig C, panels A, B and C in S1 File). In addition, Rna15 also interacted with Sqs1, Sky1 and Smb1 (Fig C, panel B in S1 File), while Rat1 made additional contacts with Prp4 and Sqs1 (Fig C, panel C in S1 File). Apart from a few non-snRNP proteins, most of these splicing factors are components of snRNP complexes. This included Prp19, which is a component of the NineTeen complex (NTC) that functions during catalytic activation of the spliceosome [39–41] and Prp43, an RNA helicase, which is involved in removing U2, U5, and U6 snRNPs from the post-splicing lariat-intron ribonucleoprotein complex [49]. Most of the splicing factors identified here are not required early in the splicing process, but after the recruitment of U1 and U2 snRNPs. Prp19 and Snu114 for example, are required for the first transesterification reaction [50–51], while Brr2 is required for disruption of U4/U6 base pairing in pre-catalytic spliceosome [52].
Splicing factor Prp19 interacts with multiple factors linked to transcription
We selected Prp19 and Prp43 for further analyses because they also exhibited interaction with TFIIB [21]. Thus, to explore the significance of the Prp19 and Prp43 interactions in transcription and cotranscriptional RNA processing in more detail, we performed reciprocal purification experiments. We epitope-tagged Prp19 and Prp43 and performed affinity purification from chromatin and soluble cellular fractions. Mass spectrometric analysis produced some rather surprising results. While no significant interaction with the transcription machinery was detected for Prp43 (Fig D in S1 File), Prp19 showed robust, statistically significant interactions with RNAPII (Fig 1B), TFIID (Fig 1C) and the CPF termination complex (Fig 1D). A related unexpected finding was evidence for a strong interaction of Prp19 with the RSC chromatin remodeling complex (Fig 1E).
The splicing factors are cotranscriptionally recruited by the CTD of RNAPII [53–55]. There are also reports of interaction between components of the Prp19 containing NTC with RNAPII [43]. We therefore examined the interaction of Prp19 with RNAPII subunits. Our results showed strong, stable interaction of Prp19 with at least nine subunits of RNAPII: Rpb1, Rpb2, Rpb3, Rpb4, Rpb5, Rpb7, Rpb8, Rpb9 and Rpb10 (Fig 1B). The high NSAF values indicate that the interaction of Prp19 with RNAPII subunits is stronger than that of any of the termination factors described above. Next, we assessed the representation of GTFs in the purified Prp19 preparations. Although several GTFs including TFIIB were detected, TFIID subunits, particularly Taf3, Taf5, Taf6, Taf12 and Taf14, exhibited a statistically significant enrichment in chromatin derived preparation (Fig 1C). The TFIID interaction may have implications in both transcription and splicing. We also looked for the presence of termination factors in the Prp19 preparation. Although Prp19 was detected in the chromatin derived preparations of CF1, CPF and Rat1 termination complexes, only CPF subunits were recorded with a stable, statistically significant presence in the affinity purified chromatin Prp19 preparation (Fig 1D). The CPF subunits Glc7, Cft1, Cft2, Ysh1, Mpe1, Fip1, Pti1 and Pfs2 were consistently detected in chromatin-linked Prp19 preparation (Fig 1D). The CF1 and Rat1 subunits were also present in Prp19 preparation, but the NSAF values were statistically not significant. Probably the interaction of Prp19 with CF1 and Rat1 complexes is not as strong as with CPF complex.
The most notable of the Prp19 interactions, however, was with RSC chromatin remodeling complex. Prp19 exhibited robust interaction with almost all subunits of the RSC complex. The RSC subunits Sth1, RSC2, RSC3, RSC4, RSC6, RSC8, RSC9, RSC58 were highly enriched in the chromatin-derived Prp19 preparation (Fig 1E). RSC complex is especially critical for initiation of transcription by virtue of its role in remodeling +1 nucleosome [56–57]. The complex has also been implicated in elongation of transcription [58]. These interactions were observed exclusively in the chromatin fraction. Taken together, these findings suggested that Prp19 may play a previously undiscovered role in the initiation and termination steps of transcription.
We verified some of the interactions of Prp19 by an alternative approach. The cell lysate from Prp19-Myc tagged strain was separated into the chromatin and soluble fractions as described above and immunoprecipitation was performed using anti-Myc antibodies. We then examined the presence of three proteins: RNAPII subunit Rpb3, TFIID subunit Taf14 and RSC subunit Snf4 in the immunoprecipitated fraction by Western blot approach. We could detect the presence of Rpb3, Taf14 and Snf4 in the chromatin fraction (Fig E in S1 File).
Evidence of Prp19 interaction with the RNA export complex TREX
In humans, multiple Prp19-containing complexes (NTC/Prp19C) have been reported [37–41]. In contrast, only one NTC/Prp19C containing nine core subunits; Prp19, Cef1, Snt309, Prp46, Syf1, Syf2, Syf3/Clf1, Isy1 and Ntc20, have been identified in budding yeast [59–60]. Except for Prp19, we could not detect any of the eight subunits of previously reported yeast NTC in our chromatin-eluted Prp19 preparation. This may be due to the presence of multiple Prp19-containing complexes in yeast like in humans. Clearly, in the chromatin environment, Prp19 is in a different interactome, distinct from the nine-subunit structure present in spliceosomes. The Prp19 complex also interacts with subunits of TREX complex in yeast [43]. We therefore looked for the presence of TREX complex subunits in the affinity purified Prp19 preparations. Two subunits of the TREX complex, Hpr1 and Thp2, were consistently detected with high confidence in the chromatin derived Prp19 preparation (Fig 1F).
These results suggest that Prp19 may interact with multiple protein partners depending on its functional state. It is also possible that the interaction of Prp19 with other eight reported subunits of yeast NTC and subunits of TREX complex is not stable enough to withstand high ionic strength used to elute the complex from chromatin.
Prp19 interaction with RNAPII, TFIID and TREX complex is not mediated by DNA or RNA
Our findings that Prp19 interacts with RNAPII, TFIID, the CPF complex, the TREX complex, and the RSC complex specifically in the chromatin context led us to speculate that these interactions might be indirect, potentially mediated by the template DNA or the transcribing mRNA. To investigate whether these interactions were dependent on nucleic acids, we subjected chromatin eluate to micrococcal nuclease (MNase) digestion before performing affinity chromatography and mass spectrometry [21]. MNase digests chromatin by cleaving exposed DNA between nucleosomes and RNA, thereby releasing any protein-DNA or protein-RNA complexes.
The red boxes indicate that the interaction of Prp19 with the protein was completely abolished in the presence of MNase, while yellow boxes indicate that interaction of the protein with Prp19 was compromised in the presence of MNase. White boxes depict the interaction being completely unaffected by MNase.
After MNase digestion, we observed that the interaction between Prp19 and most of the subunits of RNAPII, TFIID, and the TREX complex remained largely unaffected (Table 1A, 1B, 1D and 1E). This suggested that Prp19 directly associates with these complexes independent of any intermediary nucleic acids. Notably, however, the interaction with specific subunits, such as Rpb10 of RNAPII and Taf3 of TFIID, was completely abolished (Table 1A and 1B). Additionally, interactions with Taf14 and Hpr1 also showed a marked decrease (Table 1B and 1D). These observations indicated that Prp19’s association with these complexes was not generally dependent on DNA or RNA, but that certain subunits may still be partially reliant on nucleic acids for maintaining a stable interaction. In contrast, Prp19 interaction with several subunits of the CPF and RSC complexes was significantly reduced or completely lost after MNase treatment (Table 1C and 1E). This suggested that the binding of Prp19 to these complexes was, to some extent, dependent on nucleic acids, either directly or by stabilizing protein-protein interactions.
Table 1: The table shows NSAF values for all interacting partners of Prp19 shown in Fig 2, before and after MNase digestion.
To further validate the specificity of these interactions, we assessed the presence of histones in the purified Prp19-chromatin preparation. We did not find a signal for any of the four yeast histones (H2A, H2B, H3 and H4) above the no tag control in affinity purified Prp19-chromatin preparation. These results support the conclusion that Prp19 is not indiscriminately interacting with proteins bound to chromatin and that its interactions are highly selective and specific instead, reinforcing the authenticity of the identified protein-protein associations.
Prp19 has a novel role in transcription by RNAPII
The interaction of Prp19 with RNAPII, TFIID, CPF complex as well as the RSC complex gave rise to the speculation that Prp19 may have a novel role in RNAPII transcription cycle. While Syf1 and Syf2 subunits of Prp19C/NTC have been implicated in RNAPII transcription cycle at the elongation step [43–44], the direct involvement of Prp19 itself in the transcription cycle has never been demonstrated. Furthermore, Syf1 and Syf2 were not detected in our affinity purified Prp19 preparation, thereby suggesting that Prp19 is in a different interactome in chromatin environment. Thus, to explore the role of Prp19 in transcription, we performed auxin-mediated depletion of the protein in yeast cells as described in Shetty et al., [61]. The auxin-inducible degron (AID) is a powerful tool for depletion of essential proteins to study their function in vivo in non-plant eukaryotes. This method can conditionally induce the degradation of any protein by the proteasome, simply by the addition of the plant hormone auxin. Application of the AID protocol resulted in degradation of about 92% of the protein within 90 minutes of adding auxin to the medium (Fig 2A). Furthermore, the growth of Prp19-AID tagged yeast cells was adversely affected in the presence of auxin in the medium (Fig 2B). No such growth defect was observed for the isogenic wild-type strain under the same conditions (Fig 2B).
Prp19 affects transcription of both intron-containing and intron-lacking genes.(A) Western blot showing auxin-mediated depletion of AID-tagged Prp19 from yeast cells. (B) Serial dilution plating assay showing complete loss of cell viability in the presence of 500 mM of auxin. (C) Nascent mRNA level of intron-containing genes; ASC1, APE2, HPC2, and IMD4, exhibits a 15-40% decline upon auxin-mediated depletion of Prp19. (D) Nascent RNA level of intron-lacking genes; BUD3, SUR1, HEM3, and SEN1 exhibited a 25-50% reduction in the absence of Prp19. The nascent transcript level of 18S rRNA was used as the normalization control. p-values calculated by two tailed t-test. Error bars represent one unit of standard deviation based on four biological replicates.
To examine the role of Prp19 in transcription, we performed Transcription Run-On (TRO) assay in Prp19-AID-tagged strains after 90 minutes of Prp19 depletion in the presence of auxin. The TRO assay measures the level of nascent transcripts and accurately reflects the transcription of a gene. We monitored transcription of four intron-containing genes (ASC1, APE2, HPC2 and IMD4) and four intron-lacking genes (BUD3, SUR1, HEM3 and SEN1). The transcription of the four intron-containing genes decreased by about 25–40% (Fig 2C), while that of four intron-lacking genes registered a 25–50% decline upon auxin-mediated depletion of Prp19 (Fig 2D). It is possible that the decreased transcription in the absence of Prp19 is due to the reduced number of RNAPII or general transcription factor molecules in cells. We, therefore, checked for the level of RNAPII, TFIIB and TBP in the absence and presence of Prp19 in cells by Western blot approach using antibodies against Rpb3, TFIIB and TBP. We did not find any detectable decrease in the signal for Rpb3, TFIIB and TBP in the absence of Prp19 (Fig F in S1 File). These results suggest that Prp19 possibly has a more direct role in transcription of at least a subset of both intron-containing and intron-lacking genes in yeast.
Genome-wide evidence of an intron-independent role of Prp19 in gene transcription
Next, we investigated Prp19’s involvement in transcription on a genome-wide scale. To achieve this, we employed the ‘Global Run-On-Seq’ (GRO-seq) approach, which is a genome-wide, strand-specific adaptation of the traditional transcript run-on (TRO) method [62,63], The method provides a high-resolution snapshot of transcriptional activity, allowing for precise mapping of the position and density of actively transcribing RNA polymerase in a strand-specific manner. GRO-seq was performed in both wild-type and Prp19-depleted yeast cells, with three biological replicates per condition. The sequencing reads were aligned to the S. cerevisiae S288c genome (version R64-1–1) obtained from SGD. To focus our analysis on transcriptionally active genes under standard YPD growth conditions, we selected mRNAs with mean expression levels of ≥10 reads.
To investigate the differential expression of genes in wild-type and Prp19-depleted cells, we generated a mean-difference (MA) plot (Fig 3A). We used genes that were separated from their neighboring genes by at least 250 nucleotides for this analysis. This was done to remove the interfering effect of neighboring genes in our analysis. Out of these 3140 genes, 2918 had mean expression level greater than 10 and were used for further analysis. Genes exhibiting a 2-fold or greater change in expression upon Prp19 depletion are highlighted in red, while those with less than a 2-fold change, or no significant change, are shown in grey. This approach indicated that nearly 193 genes were upregulated, and 217 genes were downregulated following the depletion of Prp19 (Fig 3A). However, a considerably larger group of genes showed more subtle changes in expression, ranging from 25-50%, which is less than a 2-fold change (Fig 3A), suggesting a broader effect of Prp19 on the global gene expression landscape of yeast.
GRO-Seq analysis demonstrates genomewide alteration in transcription upon auxin-mediated depletion of Prp19 from yeast cells.(A) MA plot displaying the relationship between the log2 fold-change and the mean expression levels of genes derived from GRO-Seq reads in wild-type (WT) and Prp19-depleted cells. Genes that are significantly upregulated and downregulated are highlighted in red. The Y-axis represents the base-2 log fold-change, while the X-axis shows the normalized mean expression. Data represents the average of three biological replicates for each condition. (B) Heat maps show GRO-seq signal of the 93 downregulated intron-containing genes. The read densities are from -500 nucleotides upstream of TSS and +500 nucleotides downstream of the TES in WT and Prp19-depleted cells. All three replicates are shown here. The genes are arranged from top to bottom in descending order of relative expression. (C) Metagene plot of normalized read densities of intron-containing genes derived from heat maps in ‘B’ in WT and Prp19-depleted cells. Thick/center lines are the average normalized read densities. Upper and lower light-colored lines represent standard error. (D) Heat maps show GRO-seq signal of the 164 downregulated intron-lacking genes from -500 nucleotides upstream of TSS and +500 nucleotides downstream of the TES in WT and Prp19-depleted cells in three biological replicates. (E) Metagene plot of normalized GRO-Seq read densities of intron-lacking genes derived from heat maps in ‘D’ in WT and Prp19-depleted cells. (F) Ontological analysis of genes downregulated upon Prp19 depletion from yeast cells.
In the next step of our analysis, we focused on genes that required Prp19 for optimal expression, suggested by their downregulation in Prp19 depleted cells. Given that Prp19 is a splicing factor, and that splicing affects transcription of intron-containing genes, we categorized downregulated genes into ‘intron-containing’ and ‘intron-lacking’ groups. Of the 217 genes downregulated in the absence of Prp19, 53 were intron-containing, while 164 are intron-lacking. Intron-containing genes are strongly enriched in the downregulated set (53/217 vs 123/2198; ~ 4.4-fold enrichment; hypergeometric test p < 1e-15). Thus, Prp19 preferentially regulates intron-containing genes, but a considerable number of intron-lacking genes are also dependent on Prp19 for their optimal expression. These results suggest that Prp19-mediated regulation may not be completely dependent on splicing. Prp19 may have a splicing-dependent as well as the splicing-independent role in transcriptional regulation.
The heatmap of the 53 significantly downregulated intron-containing genes revealed a consistent reduction in transcription across the exonic regions in all three replicates in the absence of Prp19 (Fig 3B). Moreover, metaplot analysis indicated a 2–3-fold decrease in transcription of the same loci upon Prp19 depletion (Fig 3C). Notably, heatmaps of the 164 intron-lacking genes exhibited a similar reduction in transcription across coding regions in the absence of Prp19 (Fig 3D) and the metaplot for these genes further confirmed a three-fold or greater decrease in transcription following Prp19 depletion (Fig 3E).
These results collectively suggested that Prp19 is essential for the optimal transcription of a subset of both intron-containing and intron-lacking genes. The regulation of transcription of intron-containing genes by Prp19 may be dependent on its splicing function. Thus, Prp19 is not a general transcription factor like those traditionally involved in basal transcription. This distinction suggests that Prp19 might play a more specialized or context-dependent role in transcriptional regulation.
Differential gene expression evidence of a specific role of Prp19 in cell growth regulation
To explore the shared features of genes dependent on Prp19 for transcription, we searched for common structural or sequence motifs. Interestingly, however, genes whose transcription was reduced upon Prp19 depletion did not exhibit a clear pattern in terms of predicted structures or enriched sequence motifs. Additionally, Prp19-associated transcriptional reduction was not linked to the presence or absence of a TATA box in the promoter region. Both TATA-box-containing and TATA-box lacking genes exhibited Prp19-dependence, suggesting that Prp19’s role in transcription is not confined to a particular type of promoter architecture.
Ontological analysis of Prp19-dependent genes revealed that transcription of genes involved in translation, ribosomal subunit biogenesis, and ribosome assembly was particularly sensitive to Prp19 depletion (Fig 3F).
We also performed GO term analysis separately for intron-containing and intron-less downregulated genes. The GO terms related to translation and ribosome biogenesis were identified in both groups (Fig G in S1 File). This indicates that these terms are not exclusively driven by the presence of introns but reflect contributions from both intron-containing and intron-less genes. These results indicate a pivotal role of Prp19 in maintaining the transcription of genes that are essential for protein synthesis and cellular growth. In contrast, ontological analysis of genes with enhanced transcription in the absence of Prp19 revealed a strong association with processes related to cell wall and membrane biology (Fig H in S1 File). This suggested that Prp19 may exert an inhibitory effect on the transcription of gene sets involved in cell wall and membrane-related processes, further consistent with a role in fine-tuning transcriptional responses under different cellular conditions.
Overall, these findings provided compelling evidence that Prp19 is not only a splicing factor but also plays a critical, albeit specialized, role in transcription regulation.
Prp19 affects multiple steps of RNAPII transcription cycle
To gauge a deeper understanding of the role of Prp19 in transcription, we investigated its involvement in different steps of RNAPII transcription. Since proteomic analysis uncovered interaction of Prp19 with TFIID and the RSC complex, we first examined if it is involved in the assembly of preinitiation complex (PIC). To this end, we monitored the recruitment of TFIIB and the TFIID subunit TBP to the promoter regions of three intron-containing and three intron-lacking genes in the presence and absence of Prp19. Recruitment was assessed by chromatin immunoprecipitation (ChIP) approach using antibodies against the TFIIB and TBP and PCR using primer pairs shown in Fig 4A and 4C. TBP occupancy at the promoters of both gene categories decreased by approximately 65–80% in the absence of Prp19 (Fig 4B and 4D). The TFIIB promoter occupancy similarly exhibited nearly 40–70% decline upon depletion of Prp19 (Fig 4B and 4D). These results demonstrated the role of Prp19 in the assembly of PIC for both intron-containing and intron-lacking genes. Next, we checked if Prp19 is required for elongation of transcription. The transcription of all tested genes did not exhibit any significant change in the presence of 6-azuracil upon depletion of Prp19 from cells (Fig I in S1 File). Thus, Prp19 may not be affecting the elongation step of transcription of at least the intron-containing and intron-less genes tested in our analysis. Further experimentation, however, is needed to clarify the role of Prp19 in elongation.
Promoter occupancy of TATA-binding protein (TBP) and TFIIB is adversely affected in the absence of Prp19.(A) Schematic depiction of an intron-containing gene showing the position of the primer pairs 1 and 2 used in ChIP assay. (B) The intron-containing genes; APE2, ASC1, and IMD4 display a statistically significant decrease in promoter occupancy of TBP and TFIIB upon auxin-mediated depletion of Prp19 from cells. (C) Schematic depiction of an intron-lacking gene showing the position of the primer pairs 1 and 2 used in ChIP assay. (D) The intron-lacking genes; SEN1, HEM3, and SUR1 exhibit a decreased occupancy of TBP and TFIIB at the promoter region in the absence of Prp19. The Input signal, representing DNA prior to immunoprecipitation, was used as normalization control. ChIP results presented here represent the average of three biological and five technical replicates. p-values calculated by two tailed t-test indicate significant enrichment of the splicing factors factor in chromatin fraction relative to soluble fraction. One asterisk () signifies a p-value equal to or smaller than 0.05 (p ≤ 0.05); two asterisks () signify a p-value equal to or smaller than 0.01 (p ≤ 0.01); while three asterisks () signify a p-value equal to or smaller than 0.01 (p ≤ 0.001). Error bars represent one unit of standard deviation based on four biological replicates.
Since Prp19 exhibited strong interaction with CPF termination complex, we next examined if it has a role in termination step of transcription. We performed TRO assays in the presence and absence of Prp19 in cells. The TRO assay was performed for four intron-containing and four intron-lacking genes (Fig 5). When termination is defective, the TRO signal is detected downstream of the poly(A) termination site (Fig 5A). Strand-specific TRO analysis revealed that in all eight genes, there was a strong polymerase signal in the coding region before the poly(A) site in the presence of Prp19 in cells (Fig 5B and 5C, black bars). There was, however, a strongly reduced or undetectable polymerase signal in the region downstream of the poly(A) site (Fig 5B and 5C, black bars). In Prp19-depleted cells, though there was no difference in the TRO signal upstream of the poly(A) site compared to that in the presence of Prp19, there was a dramatic increase in TRO signal in the downstream regions of all eight genes (Fig 5B and 5C, white bars). This is the result of failure to read the termination signals efficiently in the absence of Prp19, and continued transcription of downstream regions, which is the hallmark termination defect.
TRO shows a transcription termination defect for both intron-containing and intron-lacking genes in the absence of Prp19.(A) Schematic depiction of a gene showing the actively transcribing RNA polymerase II and positions of primers used for RT-PCR following TRO procedure. Primers are depicted as dash; primer within the gene body is primer GB whereas primers RT1-4 are downstream of the terminator region. (B) Transcription termination defect detected as read-through of the TRO signal beyond the 3’ end for intron-containing genes; APE2, ASC1, HPC2, and IMD4, upon auxin-mediated depletion of Prp19. (C) Transcription termination defect detected as read-through of the TRO signal beyond the 3’ end for intron-lacking genes; BUD3, HEM3, SEN1, and SUR1, upon auxin-mediated depletion of Prp19. Results shown here are from three biological replicates. RT-PCR signal is represented as nascent mRNA signal compared to 18S control. p-values are a result of a standard t-test. Error bars represent standard deviation.
These results provided strong evidence that Prp19 regulates transcription by targeting multiple steps of transcription cycle. It affects assembly of PIC as well as termination step of transcription of at least a subset of intron-containing and intron-less genes in budding yeast. Prp19 may be playing a direct role in transcription of RNAPII-transcribed genes, or it may be affecting transcription indirectly. To distinguish between these possibilities, we performed Prp19-ChIP for three intron-containing and three intron-lacking genes, which showed reduced transcription upon auxin-mediated depletion of Prp19. We reasoned that if Prp19 is directly involved in transcription initiation, it would be recruited to the promoter region of these genes. The ChIP-PCR was performed using primer pairs shown in Fig 6A and 6C. The ChIP results revealed that Prp19 crosslinked to the promoter, coding region as well as the terminator region of all six genes under investigation (Fig 6B and 6D). Furthermore, there was no ChIP signal for Prp19 in the intergenic region, thereby suggesting that it is not indiscriminately binding to any region in the genome. Together, these findings demonstrate that Prp19 play a role in the transcription of both intron-containing and intron-lacking genes in yeast.
Prp19 is recruited to the promoter, terminator and coding region of both intron-containing and intron-lacking genes.(A) Schematic depiction of an intron-containing gene showing the position of the ChIP primer pairs in the open reading frame, promoter, terminator, intergenic upstream (Int-Up) and intergenic downstream (Int-Dn) regions of the gene. (B) Intron-containing genes IMD4, ASC1, and APE2 show a moderate increase in Prp19 occupancy at the promoter and terminator regions compared to the body of the gene. (C) Schematic depiction of an intron-lacking gene showing the position of the primer pairs used in ChIP assay. (D) The intron-lacking genes BUD3, HEM3, and SUR1 show an increase in Prp19 occupancy at the promoter region as compared to the body of the gene. The Input signal, representing DNA prior to immunoprecipitation, was used as normalization control. ChIP results presented here represent the average of three biological and five technical replicates. p-values calculated by two tailed t-test indicate significant enrichment of the splicing factors factor in chromatin fraction relative to soluble fraction. One asterisk () signifies a p-value equal to or smaller than 0.05 (p ≤ 0.05); two asterisks () signify a p-value equal to or smaller than 0.01 (p ≤ 0.01); while three asterisks () signify a p-value equal to or smaller than 0.01 (p ≤ 0.001). Error bars represent one unit of standard deviation based on four biological replicates.
Discussion
The classical view of transcription and cotranscriptional RNA processing is that factors involved in these processes have dedicated step-specific functions. Transcription initiation and termination factors are seen as key to the beginning and end of RNA synthesis respectively, while splicing factors are traditionally confined to their role in intron removal during RNA maturation. A number of studies, however, have increasingly challenged this dogma, revealing that many of these factors participate in broader networks that integrate transcription and RNA processing. A growing body of evidence suggests that transcription and RNA processing are not independent, temporally separate events but are intricately coupled through a network of interactions among a variety of accessory proteins including RNA-binding proteins [2,26,27,47,48,64–68]. Notably, splicing factors have been shown to influence transcription, and transcription factors have been found to modulate splicing, blurring the boundaries between these processes [28,29,34,43,44,69–72]. Here, we present data that further integrate transcription and splicing, showing that the splicing factor Prp19, a key player in spliceosome assembly, also plays a role in transcription.
Although Prp19 per se has not been directly implicated in transcription, the NTC/Prp19C has been shown to affect transcription in yeast. Syf1 and Syf2, two subunits of NTC/Prp19C, have been linked to the elongation step of transcription [43–44]. Here we provide multiple lines of evidence for a novel role of Prp19 in transcription of at least a subset of RNAPII-transcribed genes. First, Prp19 interacts with the TFIID, CPF complex and the RSC complex exclusively in the context of chromatin, which is the site of RNAPII transcription (Fig 1). The possibility of these interactions being indirect, bridged by RNAPII, however, cannot be ruled out. Second, auxin-mediated depletion of Prp19 from yeast cells adversely affected the transcription of a subset of both the intron-containing as well as intron-lacking genes (Figs 2 and 3). Third, the recruitment of TFIID and TFIIB to promoters of selected genes was compromised in the absence of Prp19 (Fig 4). Fourth, all eight tested genes exhibited a termination defect upon auxin-mediated depletion of Prp19 (Fig 5). Fifth, Prp19 crosslinked to the promoter, coding region, and the 3’ end of selected genes, with a slight peak at the promoter region (Fig 6). Taken together, these results demonstrate a novel, possibly a direct role of Prp19 in transcription by RNAPII.
Prp19 affected transcription of both intron-containing and intron-lacking genes, thereby underscoring that the factor may have both the splicing-dependent as well as splicing-independent roles in transcription. The possibility of Prp19 being a transcription factor with additional function in splicing cannot be ruled out. Prp19, however, is not an essential transcription factor as it is required for transcription of only a subset of genes, and transcription of the affected genes is reduced but is not completely abrogated in the Prp19-depleted cells. Our analysis also revealed a subset of genes that exhibited enhanced transcription in the absence of Prp19, suggesting that Prp19 has an inhibitory influence as well on transcription. Whether Prp19 is directly or indirectly inhibiting transcription of these genes needs further investigation. The association between Prp19 and processes like ribosome biogenesis further highlights its potential role in coordinating the transcriptional and translational machinery necessary for cell growth and function. This multi-functionality of Prp19 underscores the importance of moonlighting factors in cellular processes and highlights a novel aspect of transcriptional regulation that warrants further exploration.
Given its involvement in various stages of gene expression including transcription, splicing and mRNA export, we propose that Prp19 may serve as a crucial integrative hub or scaffolding protein, linking transcription with cotranscriptional splicing and post-transcriptional processes such as mRNA export and degradation (Fig 7). Further research into the dynamic roles of Prp19 and related complexes will likely reveal new insights into the integration of transcriptional and post-transcriptional regulation.
A model showing pleiotropic role of Prp19 in transcription and splicing.Prp19 role in splicing, elongation of transcription and RNA trafficking is already known. Here we show a novel role of Prp19 in the initiation as well as termination steps of transcription.
Materials and methods
Yeast strains and primers
All yeast strains used in this study are listed in Table A in S1 File. All PCR primers used in this study are listed in Table B in S1 File.
Cell culture
A 5 ml culture was started in yeast-peptone-dextrose (YPD) medium using colonies from a freshly streaked plate. The culture was grown overnight at 30°C with constant shaking at 250 rpm. All Saccharomyces cerevisiae cell cultures were grown in YPD medium unless otherwise stated. All strains were grown at 30°C and 250 rpm. The next morning, cultures grown overnight were diluted 1:100 in YPD broth and allowed to grow at 30°C with constant shaking until the desired A600 was reached. The cells were then processed accordingly for each experiment.
Cell culture for auxin-induced degradation of Prp19
The starter culture of Prp19-AID-tagged strain was grown in 5 mL of YPD medium and grown overnight at 30°C and 250 rpm in an orbital shaker. Next morning, the overnight grown culture was diluted 1:100 in appropriate medium and allowed to grow till A600 reached 0.3–0.8. At this point, one-half of the culture was treated with auxin (final concentration 500 μM) and the other with DMSO (control). Both cultures were grown for another 90 minutes. Cells were collected by centrifugation and used for RT-PCR, TRO or ChIP analysis.
Auxin plating assay
Cells were grown overnight in a 5 mL liquid YPD medium as previously described. Next day, the 5 mL culture was diluted 1:100 in YPD, and cells were allowed to grow until A600 reached ~0.4-0.6. An aliquot of cell culture was transferred to a well of 96-well plate and serially diluted (1:10, 1:100, 1:1,000, 1:10,000, 1:100,000). The dilution procedure was performed for wildtype cells and cells harboring AID-tagged Prp19. The serially diluted cells were plated on YPD or YPD + 500 μM auxin plates using a sterile prong frogger. The plates were incubated at 30°C. Images of plates were taken each day until the growing colonies reached saturation.
Purification of termination complexes
The CPF, CF1 and Rat1 complexes were purified following the protocol shown in Fig 1A from strains harboring Myc-tagged Ssu72, HA-tagged Rna15 and HA-tagged Rat1 respectively (Fig 1B). Yeast cells from 10 L of exponentially growing liquid culture were lysed, and the lysate was separated into soluble and chromatin fractions by differential centrifugation as described in [21]. The chromatin and soluble fractions were subjected to affinity chromatography on anti-HA or anti-MYC magnetic beads. 100 μL of the bead slurry was transferred to a 1.5 ml microcentrifuge tube and placed on a magnetic rack for 30 seconds to allow for the beads to settle along the magnet. The supernatant was removed, and the beads were washed with wash buffer [25 mM tris-acetate pH 7.8, 5 mM DTT, 1 mM MgCl2, 100 mM potassium acetate, and 0.05% Triton X-100] three times to allow equilibration of beads. The chromatin or soluble fraction was added to the buffer equilibrated beads and mixed gently. Protein solution (chromatin or soluble fraction) was allowed to bind to the affinity beads for 3 hours at 4°C. Binding was performed by gentle shaking the bead and protein solution on a nutator. After binding, the supernatant was carefully removed, and beads were washed three times with wash buffer. Bound proteins were eluted with 250 μL of elution buffer (Tris-HCl pH 6.8, 60 mM, 10% glycerol, 2% SDS and 500 mM β-mercaptoethanol). Elution was performed at room temperature for 30 minutes on a benchtop nutator. The eluted proteins were separated from the beads by placing the tubes on a magnetic rack. The eluent was transferred to a new tube and stored at −80°C.
MNase digestion
Micrococcal Nuclease (MNase) digests both DNA and RNA. MNase digestion was performed before affinity purification. The chromatin eluted samples were digested with 20,000 units of MNase in the presence of 5 mM calcium chloride in 1 mL of sample volume at 37°C for 30 minutes.
Proteomic analysis
Analysis of the mass spectrometry data from purified protein preparations was done essentially as described in [21]. The mass spectrometry data were compiled into Scaffold files. The Scaffold program display was set to protein name and species (S. cerevisiae), UniProt accession number, alternate protein name identification, molecular weight, and, importantly, the normalized total spectra (spectral counts). Spectral counts greater than 95% were included in our analysis. The protein threshold was set to 0.1% false discovery rate (FDR), the minimum number of peptides was set to 1, and the peptide threshold was set to 0.1% FDR. Spectral counts for each protein in tagged and untagged/control replicate samples were divided by their molecular weight to produce the spectral abundance factor (SAF), as described in [73–74]. The average SAF value for untagged replicates was subtracted from each tagged replicate SAF value. The SAF values were then normalized against the SAF of the bait/tagged proteins Ssu72, Rna15, and Rat1 to generate the normalized spectral abundance factor (NSAF). Finally, the NSAF values from three biological replicates were averaged to generate a mean NSAF value for each interactor. Following this protocol, the NSAF values of each Ssu72, Rna15 and Rat1 interacting protein in the soluble and chromatin fraction were calculated and tested for significant enrichment by a two-tailed standard t-test. A p-value of 0.05 or less indicated a significant difference between fractions or samples, with standard error accounting for the variability across replicates. The authenticity of the soluble and chromatin fractions was verified using marker proteins, α-tubulin for soluble fraction, and histones for the chromatin fraction of the cell lysate. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD061187 and 10.6019/PXD061187.
Auxin-mediated depletion of Prp19
The overnight grown cultures were diluted to 1:100 in 100 mL YPD and grown to an A600 of 0.3-0.5 as described above. Cell cultures were incubated with 500 μM (final concentration) auxin (Tocris - 6834) for 90 minutes in a 30°C incubator with gentle shaking. Treated cells were then used for further analysis.
Transcription run-on (TRO) assay
The overnight grown cultures were diluted to 1:100 in 100 mL YPD and grown to an A600 of 0.8 as described above. The 4-thiouracil was now added to the culture from 2 M stock to a final concentration of 5 mM. The incorporation of 4-thiouracil into nascent RNA was allowed for exactly 5 minutes at 30^O^C in an orbital shaker. The cell culture was then transferred to 50 mL conical tubes and centrifuged at 3000xg for 2 mins at 4^O^C. The cell pellet was washed with 20 mL of cold 1xPBS, and washed cell pellet is transferred to a 1.5 mL microfuge tube. The cell pellet was flash frozen with liquid nitrogen and stored in a -80^O^C freezer. At this stage, samples can be stored overnight, and the protocol is resumed next day.
Next day, samples were removed from storage and thawed on ice. Cells were spun down in a microcentrifuge at 1,100xg for 5 minutes at 4°C. The cell pellet is quickly resuspended in 500 μL of phenol (pH 4.5). An equal volume of AES (50 mM NaOAc pH 5.3, 10 mM EDTA pH 8, and 1% SDS) buffer is added, and the sample is incubated in a 65°C water bath for 5 minutes. During the 5-minute incubation period the sample contents are vortexed for 10 seconds once every minute. The samples were then incubated on ice for 5 minutes followed by addition of 200 μL of chloroform to each sample. The samples were mixed vigorously on a vortexer for 30 seconds, incubated at room temperature for 2 minutes followed by centrifugation at 14,220xg for 5 minutes at 4 °C in a microcentrifuge. The upper aqueous phase was transferred to a new tube and ethanol precipitated for 10 minutes in the presence of 2 μL GlycoBlue before spinning down precipitated RNA. The RNA pellet was washed with 750 μL of ice-cold 75% ethanol and centrifuged at 14,220 x g for 5 minutes at 4°C. The supernatant was carefully removed, and the pellet was air dried for 5–10 minutes at room temperature. It was important not to let the RNA pellet dry completely as this will greatly decrease its solubility in the next step. Finally, the RNA pellet was resuspended in 100 μL DEPC-H_2_O.
The concentration of RNA was measured using a Nanodrop. The total concentration of RNA for the following steps was adjusted to 2 mg/ml and aliquots of 200 μg of total RNA were made for the remaining processing steps. The 200 μg RNA aliquots were heated for 10 minutes in the 65°C water bath to remove any secondary RNA structures. RNA was then biotinylated by adding 650 μL of DEPC water, 100 μL of the biotinylation buffer (100 mM Tris-HCl pH 7.5 and 10 mM EDTA) and 150 μL of HPDP-biotin (stock 1 mg/ml). The RNA solution was mixed thoroughly and incubated at room temperature in dark for three hours on the nutator in a 2 mL tube. After incubation, an equal volume of chloroform was added to the tube and mixed thoroughly by a vortexer before spinning at 13,000 x g for 5 mins in a 4°C microcentrifuge. The supernatant was transferred to a new 1.5 mL microfuge tube and ethanol precipitated using Glycol-blue as described above. The pellet was resuspended in 100 μL of DEPC-treated water. The RNA suspension is then purified using a Qiagen RNeasy kit following manufacturer’s protocol. The final volume following purification over the RNeasy kit was 200 μL. The concentration of RNA was measured using Nanodrop. The yield of RNA at this stage is in the range of 100–500 μg. RNA at this stage can be stored at -80 °C for several months.
The RNA sample was removed from storage and thawed on ice. The following buffers were added in this order: 25μL 10X NaTM buffer (0.1M Tris-HCl pH 7.0, 2 M NaCl, and 250 mM MgCl2), 25μL NaPi buffer pH 6.8 (0.5 M NaH2PO4 and 0.5M NaHPO4), and 2.5 μL of 10% SDS. The contents were mixed thoroughly and spun down quickly to remove liquid from the sides. In a new 1.5 mL tube, 100 μL slurry of streptavidin beads was added and placed on a magnetic rack. The storage buffer was carefully removed from the magnetic beads. The streptavidin beads were washed with 200μL of bead buffer (200 μL NaTM buffer, 200 μL NaPi buffer, 20 μL 10%SDS, and 1.58 mL ddH2O) followed by a brief vortexing and spinning at max speed for 5 seconds. The tube was placed on the magnetic rack to allow beads to settle to the magnet before removing the buffer. The beads were then blocked by adding 200 μL of bead buffer, 10 μL 20mg/mL glycogen, and 1.25 μL 10 mg/mL E. Coli tRNA and placed on the nutator at room temperature for 20 minutes. The tubes were then placed on the magnetic rack to remove the blocking buffer and washed once with bead buffer as previously described. The RNA suspension was added to the beads after heating at 65°C for 10 minutes in a water bath. After combining the RNA suspension with the magnetic streptavidin beads, the sample was incubated at room temperature on a nutator for 90 minutes to enable bonding of biotinylated RNA to streptavidin beads. The tube was placed on a magnetic rack and the supernatant was removed. The beads were then washed three times with the bead buffer as previously described to remove any unbound RNA. To elute RNA from the streptavidin beads, 0.5 mL of Trizol was added to the tube. The contents were mixed thoroughly and incubated at room temperature for 5 minutes. 100 μL of chloroform was added to the tube and mixed vigorously on a vortexer before being incubated at room temperature for 10 minutes. A chloroform extraction is performed by centrifugation in a tabletop centrifuge at 14,220 x g for 10 minutes at 4°C. The upper aqueous phase containing RNA is carefully transferred to a 1.5 mL microcentrifuge tube. The RNA is then ethanol precipitated in the presence of 2μL of glycoblue. The supernatant is removed, and the RNA pellet should be visible at the bottom of the tube. It was crucial at this stage to carefully remove any residual buffer as the RNA pellet is flimsy and easily detaches from the wall of the microcentrifuge tube. The pellet was washed with 1 mL of ice-cold 75% ethanol. The supernatant was removed following the wash step and the RNA pellet was air-dried for 5–10 minutes at room temperature. Finally, the pellet was resuspended in 22 μL of DEPC-H_2_O and was ready to be used for subsequent RT-PCR or GRO-seq analysis.
GRO-Seq
GRO-seq was performed essentially as described in [45] except that 4-thiouracil was used instead of Br-dUTP. 4-thiouracil labeling was performed as described in TRO assay above. Nascent, isolated RNA obtained using GRO-Seq was obtained from three biological replicates. The GRO-Seq data have been deposited in the NCBI database. The NCBI Geo accession number is GSE294484.
GRO-Seq analysis
Raw GRO-Seq reads were first assessed for quality using FastQC and were then aligned to the Saccharomyces cerevisiae S288C genome (SGD version R64-1–1) using STAR (v2.7.11a) with the following parameters: maximum intron size of 2,000 nucleotides, a minimum overlap for paired-end alignment of 10 and sorted BAM alignment format. Files were then sorted by co-ordinates using STAR. Alignment quality was evaluated using SAM tools (v1.9), and BAM files were indexed for downstream analysis.
Count matrix was generated using featuresCount–countReadPairs -s2 -T6 -texon (subread-2.06). The counts obtained were processed in R/Bioconductor as described in [75–76]. Differential expression analysis was performed using DESeq2. It identified significantly differentially expressed genes with an adjusted p-value < 0.05 and a log₂ fold-change of greater than 2.
Bamcoverage (version) was used to generate bigwig files, which were imported into Integrated Genome Browser (IGV) for gene browser track visualization. The subsequent data in the bigwig file, representing nascent transcription levels from GRO-seq, was selectively enriched for the genomic regions specified in the BED files using the computeMatrix function (Galaxy Version 3.3.0.0.0). A heatmap was generated using plotHeatmap (Galaxy Version 3.3.0.0.1) to visualize changes in transcriptional activity across these regions under wild-type conditions and Prp19-depleted conditions (+auxin). Additionally, plotProfile (Galaxy Version 3.3.0.0.0) was used to generate metagene plots, allowing for the comparative analysis of nascent RNA distributions across annotated genomic regions.
The following BED files were used in this study:
93 intron-containing genes with transcription start site (TSS) and transcription end site (TES) coordinates.302 non-intronic genes with TSS and TES coordinates.
Genes were classified as TATA-containing or TATA-less based on a curated dataset of yeast genes characterized by [77]. Gene ontology analysis was performed using clusterProfiler with org.Sc.sgd.db annotation.
Chromatin Immunoprecipitation (ChIP)
Cells were grown in appropriate medium as described above in 100 mL culture. Once the cells reached an A600 ~ 0.7-0.8 crosslinking was performed with 1% formaldehyde (2.7 ml stock/100 mL cell culture) for 20 minutes at room temperature with vigorous shaking. The reaction was stopped by addition of glycine to a final concentration 125 mM, and cultures were shaken vigorously for an additional 5 minutes at room temperature. The cell culture was transferred to a 50 mL tube and spun for 5 minutes at 3,000 rpm in a Sorvall RC6Plus centrifuge. The supernatant was discarded, and the cell pellet was washed once with 10 mL of ice-cold 1xTBS buffer containing 1% Triton X-100, and twice with 1xTBS buffer only. The cell pellet was resuspended in 800 μL of cold FA-lysis buffer (50 mM HEPES-KOH pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X, 0.1% sodium deoxycholate, 1 mM PMSF, and 0.1% SDS). The cell suspension was then frozen with liquid nitrogen and stored at -80°C.
Next, cells were thawed and approximately 400 μL of acid-washed glass beads were added to each tube. Cells were lysed by vigorous shaking at 4°C for 40 minutes. The cell lysate was collected by puncturing the bottom of the tube with a red hot 22-gauge needle and collecting the lysate in a 15 mL tube by spinning at 1,000 rpm at 4°C in a Sorvall RC6Plus centrifuge. The filtrate was then transferred into a 1.5 ml microfuge tube and spun at 4°C for 15 minutes at the maximum speed in a microcentrifuge. The supernatant was discarded, and the crude chromatin pellet was washed with 1,000 μL of FA-lysis buffer and resuspended in 1,000 μL of FA-lysis buffer. The crude chromatin preparation obtained was diluted to 4 mL by the addition of FA-lysis buffer and 40 μL of PMSF. Chromatin was sheared by sonication with thirty to forty-five pulses (depending on gene size) of 20 seconds each with 30 second cooling after each pulse (This will result in a total sonication time of 10–15 minutes). Sonication was performed at the 30% duty cycle in a Branson digital sonifier. Following sonication, samples were centrifuged at 14,000 rpm for 15 minutes in a 4°C centrifuge. The pellet was discarded, and the supernatant was used in subsequent steps. The supernatant can be frozen in liquid nitrogen and stored at -80°C at this stage.
The sonicated chromatin was thawed and 300 μL of chromatin is used for each ChIP assay. 50 μL is stored aside as an input control. Approximately 5–10 μg of appropriate antibody-conjugated magnetic beads were added to a 1.5 mL tube (Pierce anti-c-Myc magnetic beads (88842) for Prp19; Santa Cruz Biotechnology TBP antibody (sc-74596) for TBP ChIP; BioAcademia Anti-Sua7p antibody (62–009)). The storage buffer was removed by a brief spin and 300 μL of chromatin was added to the beads. The chromatin was allowed to bind to beads for 4 hours at 4°C with gentle shaking on a nutator. The beads were washed successively with 1 mL each of FA-lysis buffer containing 0.25% SDS, FA-lysis buffer containing 500 mM NaCl and 0.25% SDS (50 mM HEPES-KOH pH 7.9, 500 mM NaCl, 1 mM EDTA, 1% Triton X, 0.1% sodium deoxycholate, 1 mM PMSF, and 0.25% SDS), ChIP wash buffer 0.25% SDS (10 mM Tris-HCl pH 7.5, 250 mM LiCl, 0.5% Triton X, 1 mM EDTA pH 8, 0.5% sodium deoxycholate, and 0.25% SDS) and 1XTE buffer. The washing steps were performed twice at room temperature with the 1.5 mL tubes being placed over the magnetic rack to remove the supernatant containing unbound chromatin. After washing, the beads were resuspended in 100 μL of ChIP elution buffer (50 mM Tris-HCl pH 7.5, 1% SDS, and 10 mM EDTA pH 8) and incubated at 65°C for 10 minutes in a water bath to elute chromatin from the magnetic beads. The 1.5 mL tubes containing the chromatin-bead mix was placed over the magnetic rack to remove the supernatant to transfer it to a new 1.5 mL tube. The elution was performed twice. There should be 200 μL of eluted chromatin after the elution steps. The chromatin eluent was incubated with 10 μg of DNase-free RNase (Worthington) for 30 minutes in a 37°C incubator. 20 μg of proteinase K and 2.5 μL10% SDS were added and incubated at 42°C for 90 minutes in a water bath. Finally, the protein-DNA crosslinks were reversed by overnight incubation at 65°C in a water bath.
Next day, samples were extracted with equal volumes of DNA phenol-chloroform at least two times followed by ethanol precipitation of DNA in the presence of carrier glycogen. The DNA pellet was resuspended in 50 μL of 1XTE and used as template for further PCR analysis.
Treatment with 6-azauracil
The overnight grown cultures were diluted to 1:100 in 100 mL YPD and grown to an A600 of 0.6 and depleted for Prp19 using auxin as described above. Cell cultures were incubated with 100 μg/ml final concentration of 6-azauracil (Millipore Sigma – A1757) for 120 minutes in a 30°C incubator shaker as described in Riles et al., [78]. RNA was isolated from cells as described in McNeil and Smith [79].
Supporting information
S1 FileTable A: Yeast Strains.Table B: Primers. Fig A: All three termination complexes in yeast interact with RNAPII subunits in the chromatin fraction. (A) Ssu72 subunit of CPF complex, (B) Rna15 subunit of CF1 complex, and (C) Rat1 subunit of Rat1 complex all associate with multiple subunits of RNAPII in the chromatin context. p-values calculated by two tailed t-test indicate significant enrichment of the splicing factors factor in chromatin fraction relative to soluble fraction. One asterisk () signifies a p-value equal to or smaller than 0.05 (p ≤ 0.05); two asterisks () signify a p-value equal to or smaller than 0.01 (p ≤ 0.01); while three asterisks () signify a p-value equal to or smaller than 0.01 (p ≤ 0.001). Error bars represent one unit of standard deviation based on four biological replicates. Fig B: All three termination complexes in yeast interact with general transcription factors in the chromatin fraction. (A) The Ssu72 subunit of CPF complex associates with subunits of TFIID and SAGA complex. (B) Rna15 subunit of CF1 complex interacts with TFIID, TFIIB and SAGA subunits. (C) Rat1 subunit of Rat1 complex associates with subunits of TFIID, TFIIB, and TFIIE in the chromatin context. p-values calculated by two tailed t-test indicate significant enrichment of the splicing factors factor in chromatin fraction relative to soluble fraction. One asterisk () signifies a p-value equal to or smaller than 0.05 (p ≤ 0.05); two asterisks () signify a p-value equal to or smaller than 0.01 (p ≤ 0.01); while three asterisks () signify a p-value equal to or smaller than 0.01 (p ≤ 0.001). Error bars represent one unit of standard deviation based on four biological replicates. Fig C: Purified termination complexes interact with splicing factors exclusively in the chromatin fraction. (A) Splicing factors interacting with the Ssu72 subunit of CPF complex. (B) Splicing factors interacting with the Rna15 subunit of CF1 complex. (C) Splicing factors interacting with the Rat1 subunit of Rat1complex. All splicing factor-termination factor interactions were observed in the chromatin context (blue bar). p-values calculated by two tailed t-test indicate significant enrichment of the splicing factors factor in chromatin fraction relative to soluble fraction. Error bars represent one unit of standard deviation based on four biological replicates. The method for calculating NSAF is described in detail in ‘Proteomic Analysis’ section of ‘Materials and Methods’. Fig D: The splicing factor Prp43 did not display any significant enriched interaction with GTFs in the chromatin fraction. Although Prp43 interacted with Taf6 and Spt15, the protein did not have significant enrichment in the chromatin as compared to soluble fraction. Error bars represent one unit of standard deviation based on four biological replicates. Fig E: IP-Western blot analysis of Prp19 showing its interaction with Snf4, Taf14 and Rpb3. Prp19 was affinity purified from chromatin fraction as described previously. Immunoprecipitated proteins were separated by SDS-PAGE and Western blot was performed using antibodies against Rpb3 subunit of RNAPII, Taf14 and Snf4-HA tag. No antibody control do not show any interaction of Prp19 with Rpb3, Snf4 or Taf14. Input is the protein eluted from chromatin fraction while IP is immunoprecipitated proteins. Fig F: The level of RNAPII, TFIIB and TBP remains unchanged upon auxin-mediated depletion of Prp19 from yeast cells. Prp19 was depleted from cells as described previously. Equal number of cells were lysed using 2xLaemmli buffer. Proteins in lysate were separated by SDS-PAGE and Western blot was performed using antibodies against Rpb3 subunit of RNAPII, TFIIB, TBP and α-tubulin. The α-tubulin is the loading control showing that an equal amount of protein was loaded in the Prp19-containing (+) and Prp19-depleted (-) cells. Fig G: Separate ontological analyses for intron-containing and intron-less genes. The analyses revealed that Prp19 has a stimulatory effect on transcription of genes linked to translation, ribosome biogenesis and RNA processing for both intron-containing and intron-less groups. The plot shows specific categories of genes that are downregulated upon auxin-mediated depletion of Prp19 from yeast cells. Fig H: Ontological analysis revealed that Prp19 has an inhibitory role in transcription of genes linked to cell wall and membrane biology. The plot shows specific categories of genes that are upregulated upon auxin-mediated depletion of Prp19 from yeast cells. Fig I: Prp19 does not affect the elongation step of transcription. To determine whether there is an elongation defect in the absence of Prp19, cells were treated with 6-azauracil in the presence and absence of Prp19 and the steady state level of mRNA of six genes were estimated. In the presence of 6-azauracil mRNA level of six genes, APE2, ASC1, HPC2, IMD4, BUD3, and HEM3 remained unchanged as compared to that in the absence of 6-azauracil irrespective of the presence (DMSO) or absence (auxin) of Prp19 in cells.(PDF)
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