SummaryProtein ADP-ribosylation is a highly dynamic post-translational modification. The rapid turnover is achieved, among others, by ADP-(ribosyl)hydrolases (ARHs), an ancient family of enzymes that reverses this modification. Recently ARHs came into focus due to their role as regulators of cellular stresses and tumor suppressors. Here we present a comprehensive structural analysis of the enzymatically active family members ARH1 and ARH3. These two enzymes have very distinct substrate requirements. Our data show that binding of the adenosine ribose moiety is highly diverged between the two enzymes, whereas the active sites harboring the distal ribose closely resemble each other. Despite this apparent similarity, we elucidate the structural basis for the selective inhibition of ARH3 by the ADP-ribose analogues ADP-HPD and arginine-ADP-ribose. Together, our biochemical and structural work provides important insights into the mode of enzyme-ligand interaction, helps to understand differences in their catalytic behavior, and provides useful tools for targeted drug design.

Transcription and co-transcriptional processes, including pre-mRNA splicing and mRNA cleavage and polyadenylation, regulate the production of mature mRNAs. The carboxyl terminal domain (CTD) of RNA polymerase (pol) II, which comprises 52 repeats of the Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 peptide, is involved in the coordination of transcription with co-transcriptional processes. The pol II CTD is dynamically modified by protein phosphorylation, which regulates recruitment of transcription and co-transcriptional factors. We have investigated whether mature mRNA levels from intron-containing protein-coding genes are related to pol II CTD phosphorylation, RNA stability, and pre-mRNA splicing and mRNA cleavage and polyadenylation efficiency. We find that genes that produce a low level of mature mRNAs are associated with relatively high phosphorylation of the pol II CTD Thr4 residue, poor RNA processing, increased chromatin association of transcripts, and shorter RNA half-life. While these poorly-processed transcripts are degraded by the nuclear RNA exosome, our results indicate that in addition to RNA half-life, chromatin association due to a low RNA processing efficiency also plays an important role in the regulation of mature mRNA levels.

Transcription and co-transcriptional processes, including pre-mRNA splicing and mRNA cleavage and polyadenylation, regulate the production of a mature mRNA. The carboxyl terminal domain (CTD) of RNA polymerase (pol) II, which comprises 52 repeats of the Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 peptide, is involved in the coordination of transcription with co-transcriptional processes. The pol II CTD is dynamically modified by protein phosphorylation, which regulates recruitment of transcription and co-transcriptional factors. We have investigated whether cytoplasmic levels of mature mRNA from intron-containing protein-coding genes can be inferred from RNA processing efficiency, CTD phosphorylation, and/or association of proteins complexes regulating RNA production. Surprisingly, we found that genes associated with efficient RNA processing have relatively low phosphorylation of the pol II CTD. In contrast, protein-coding genes that produce a low level of mature mRNA are associated with high pol II CTD phosphorylation, poor RNA processing, and increased chromatin retention. Unexpectedly, the transcripts from this subset of genes are not actively degraded by the RNA exosome. RNA exosome-regulated genes are instead characterised by Tyr1 hyperphosphorylation and Integrator recruitment around the poly(A) site. Our results indicate that in addition to the transcription level, CTD phosphorylation and RNA processing efficiency play important roles in the regulation of chromatin retention of transcripts.