# N-terminal tagging of RNA Polymerase II shapes transcriptomes more than C-terminal alterations

## TL;DR

Modifying the N-terminus of RNA Polymerase II has a bigger impact on gene expression than changing its C-terminal domain.

## Contribution

N-terminal tagging of Pol II, not CTD alterations, shapes transcriptomes and affects non-coding RNA and LLPS-related factors.

## Key findings

- Transcriptional bursting remains largely unchanged with CTD modifications.
- N-terminal tags significantly alter transcriptome-wide gene expression patterns.
- N-terminal tags correlate with changes in non-coding RNA and LLPS-related factor expression.

## Abstract

RNA polymerase II (Pol II) has a C-terminal domain (CTD) that is unstructured, consisting of a large number of heptad repeats, and whose precise function remains unclear. Here, we investigate how altering the CTD’s length and fusing it with protein tags affects transcriptional output on a genome-wide scale in mammalian cells at single-cell resolution. While transcription generally appears to occur in burst-like fashion, where RNA is predominantly made during short bursts of activity that are interspersed with periods of transcriptional silence, the CTD’s role in shaping these dynamics seems gene-dependent; global patterns of bursting appear mostly robust to CTD alterations. Introducing protein tags with defined structures to the N terminus cause transcriptome-wide effects, however. We find the type of tag to dominate characteristics of the resulting transcriptomes. This is possibly due to Pol II-interacting factors, including non-coding RNAs, whose expression correlates with the tags. Proteins involved in liquid-liquid phase separation appear prominently.

•ScRNA-seq datasets for cells expressing CTD-mutant RNA polymerase II (Pol II)•Transcriptional bursting is robust to Pol II CTD alterations•N-terminal- rather than CTD-modifications of Pol II shape transcriptomes•Altered expression of ncRNAs and LLPS-related factors due to N-terminal Pol II tags

ScRNA-seq datasets for cells expressing CTD-mutant RNA polymerase II (Pol II)

Transcriptional bursting is robust to Pol II CTD alterations

N-terminal- rather than CTD-modifications of Pol II shape transcriptomes

Altered expression of ncRNAs and LLPS-related factors due to N-terminal Pol II tags

Molecular biology; Biophysics; Transcriptomics