# DRBD3 regulates long non-coding RNA abundance and cryptic splice site selection in trypanosomes

## TL;DR

This paper shows that DRBD3, an RNA-binding protein in trypanosomes, regulates lncRNA levels and splicing, affecting both coding and non-coding gene expression.

## Contribution

The study reveals DRBD3's novel role in regulating cryptic splice sites and lncRNA abundance via PAP1 levels in trypanosomes.

## Key findings

- DRBD3 depletion upregulates ~100 lncRNAs via cryptic splice site activation.
- DRBD3 loss affects protein-coding gene processing, causing alternative trans-splicing and protein truncation.
- DRBD3 regulates splicing of RBP20 transcript and balances trans- and cis-splicing.

## Abstract

Trypanosomes are unicellular eukaryotes that rely heavily on post-transcriptional mechanisms to control gene expression. DRBD3 is an RNA-binding protein known to play important roles in mRNA processing, stability, transport and translation. It was found to associate with grumpy, a long non-coding RNA (lncRNA) recently characterized in Trypanosoma brucei. Here, we explore the role of DRBD3 in lncRNA metabolism and show that its depletion leads to the upregulation of a specific subset of approximately one hundred lncRNAs in both bloodstream and procyclic forms, likely through the activation of cryptic splice sites. The effect of DRBD3 depletion on lncRNA expression appears to be mostly indirect, and results from reduced levels of the poly(A) polymerase PAP1 following DRBD3 silencing. In addition to its impact on lncRNAs, DRBD3 loss also affects the processing of protein-coding genes, leading to alternative trans-splicing and protein truncation. Furthermore, we demonstrate that DRBD3 regulates the splicing of the newly identified intron in the transcript encoding the RNA-binding protein RBP20, and is important for maintaining the balance between trans- and cis-splicing. Our results position DRBD3 as a high-level regulatory factor that shapes the expression landscape of both coding and non-coding genes in trypanosomes.

The online version contains supplementary material available at 10.1007/s00018-025-05929-w.