Biology subjects

Akkawi, C.

Publications and source records attributed to Akkawi, C..

3 recordsLinked to original sources

bioRxiv2025

Nuclear exosome targeting complexes modulate cohesin binding and enhancer-promoter interactions in 3D

Three-dimensional long-range contacts between enhancers and promoters are thought to be largely determined by loop extrusion driven by the cohesin complex and insulator factors. However, recent evidence also suggests a role for noncoding RNAs (ncRNAs), such as enhancer-associated RNAs (eRNAs) and promoter upstream transcripts (PROMPTs), in shaping enhancer-promoter connectivity. While nuclear RNA exosome, together with targeting complexes, PAXT and NEXT, control the decay of ncRNAs, it has not yet been determined whether these complexes regulate 3D contacts. Chromatin recruitment maps of ZCCHC8 (NEXT), ZFC3H1 (PAXT) and MTR4 helicase revealed that these factors that associate with sites of enhancer-promoter interactions. Depletion of NEXT, PAXT or MTR4 induced the accumulation of ncRNAs, notably enhancer-associated RNAs (eRNAs) and promoter upstream transcripts (PROMPTs). Strikingly, this further increased cohesin levels at sites accumulating ncRNAs. Chromatin conformation capture analysis revealed that MTR4 modulates the 3D long-range contacts between enhancers with their distant TSS targets. Upon loss of MTR4, contacts at anchor points increase while intraloop contacts decrease, suggesting that MTR4 facilitates loop extrusion. These data highlight a key interplay between cohesin-mediated enhancer-promoter interactions and the regulation of ncRNAs by nuclear RNA exosome that is consistent with a role for RNA in genome folding.

molecular biology↗

bioRxiv2025

NRDE2 Interacts with an Early Transcription Elongation Complex and Widely Impacts Gene Expression

NRDE2 is a highly conserved protein implicated in post-transcriptional gene silencing in Schizosaccharomyces pombe and Caenorhabditis elegans and has been shown to modulate splicing in mammals. To explore whether NRDE2 participates in additional processes in human cells, we performed tandem affinity purification followed by proteomic analysis of NRDE2 from nuclear extracts of HEK293T and HeLa cells. Our analysis confirmed the interaction of NRDE2 with its well-characterized partner, the MTR4 helicase (MTREX), as well as with multiple splicing factors. Notably, we also identified interactions with chromatin-associated proteins involved in transcription, including the Polymerase-Associated Factor 1 (PAF1) complex and elongating forms of RNA polymerase II (RNAPII). To further investigate NRDE2 function, we conducted RNA-seq following its transient depletion. Differential expression analysis revealed that loss of NRDE2 alters the expression of thousands of genes. Consistent with earlier reports, we observed splicing defects, particularly intron retention; however, our results indicate that the impact of NRDE2 on intron retention is more extensive than previously recognized. Moreover, intron retention was frequently associated with reduced mRNA expression. Together, these findings suggest that NRDE2 associates with both the transcriptional and splicing machineries and plays a broader role in RNA processing than previously appreciated.

molecular biology↗

bioRxiv2023

PAP-gamma associates with PAXT nuclear exosome to control the abundance of PROMPT ncRNAs

Pervasive transcription of the human genome generates an abundance of RNAs that must be processed and degraded. The nuclear RNA exosome is the main RNA degradation machinery in the nucleus. However, nuclear exosome must be recruited to its substrates by targeting complexes, such as NEXT or PAXT. By proteomic analysis, we have identified additional subunits of PAXT, including many orthologs of MTREC found in S. pombe. In particular, we show that polyA polymerase gamma (PAP{gamma}) was associated with PAXT. Genome-wide mapping of the binding sites of ZFC3H1, RBM27 and PAP{gamma}, showed that PAXT is recruited to the TSS of hundreds of genes. Loss of ZFC3H1 abolished recruitment of PAXT subunits including PAP{gamma} to TSSs and concomitantly increased the abundance of PROMPTs at the same sites. Moreover, PAP{gamma}, as well as MTR4 and ZFC3H1, was implicated in the polyadenylation of PROMPTs. Our results thus provide key insights into the direct targeting of PROMPT ncRNAs by PAXT at their genomic sites.

molecular biology↗