Biology subjects

Kiernan, R.

Publications and source records attributed to Kiernan, R..

7 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↗

bioRxiv2021

NF90 Interacts with Components of RISC and Modulates Association of Ago2 with mRNA

Nuclear Factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity. Here, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3 UTRs, NF90 association was increased upon loss of MOV10 and vice versa, suggesting that the two proteins may compete for the binding of common target mRNAs. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased. These findings suggest a role for NF90 in the regulation of RISC-mediated silencing which stabilizes target mRNAs, such as VEGF, during cancer-induced hypoxia.

molecular biology↗

bioRxiv2020

PAF1 facilitates RNA polymerase II ubiquitination by the Elongin A complex through phosphorylation by CDK12

The conserved Polymerase-Associated Factor 1 complex (PAF1C) regulates all stages of the RNA polymerase II (RNAPII) transcription cycle from the promoter to the 3 end formation site of mRNA encoding genes and has been linked to numerous transcription related processes. Here, we show that PAF1 interacts with Elongin A, a transcription elongation factor as well as a component of a cullin-RING ligase that targets stalled RNAPII for ubiquitination and proteasome-dependent degradation in response to DNA damage or other stresses. We show that, in absence of any induced stress, PAF1 physically interacts with the E3 ubiquitin ligase form of the Elongin A complex and facilitates ubiquitination of RNAPII. We demonstrate that this ubiquitination is dependent of the Ser2 phosphorylation of the RNAPII carboxy-terminal domain (CTD) by CDK12. Our findings highlight a novel unexpected role of PAF1-CDK12 in RNAPII transcription cycle, raising the possibility that the Elongin A ubiquitin ligase plays a role in normal transcription process, and suggest a transcription surveillance mechanism ready to degrade RNAPII if needed.

cell biology↗

bioRxiv2020

Chromatin-associated MRN complex protects highly transcribing genes from genomic instability

The MRN-MDC1 complex plays a central role in the DNA damage response (DDR) and repair. Using Proteomics of Isolated Chromatin Fragments (PICh), we identified DDR factors, such as MDC1, among those that become highly associated with a genomic locus upon transcriptional activation. Purification of endogenous MDC1, in the absence of exogenous DNA damage, revealed its interaction with factors involved in gene expression and co-transcriptional RNA processing, in addition to DDR factors. ChIP-seq analysis showed that MDC1 interacting factors, MRE11 and NBS1 subunits of MRN, were co-localized throughout the genome and notably at TSSs and gene bodies of actively transcribing genes. Blockade of transcriptional elongation showed that binding of MRN was dependent on the RNAPII transcriptional complex rather than transcription per se. Depletion of MRN increased RNAPII abundance at TSSs and gene bodies of MRE11/NBS1-bound genes. Prolonged exposure of cells to either MRE11- or NBS1-depletion led to single nucleotide polymorphism formation across actively transcribing, MRE11 or NBS1 target genes. These data support a model by which association of the MRN complex with the transcriptional machinery constitutively scans active genes for transcription-induced DNA damage to preserve the integrity of the coding genome.

molecular biology↗

bioRxiv2020

NF90 Modulates Processing of a Subset of Human Pri-miRNAs

MicroRNAs are predicted to regulate the expression of more than 60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The dsRNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of pri-miRNAs. Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 22 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to acquisition of NF90 association, as determined by RNA EMSA. NF90-bound and modulated pri-miRNAs are embedded in introns of host genes and expression of several host genes is concomitantly modulated. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.

molecular biology↗