Title: Structural and Biochemical Characterizations of the Symplekin-Ssu72-CTD Complex in Pre-mRNA 3' end Processing
Description: RNA polymerase II (RNAP II) transcribes essentially all messenger RNAs (mRNAs) in eukaryotes. The C-terminal domain (CTD) of its largest subunit contains consensus heptad repeats Y₁S₂P₃T₄S₅P₆S₇. Dynamic post-translational modifications of the CTD regulate RNAP II transcriptional activity and also facilitate transcription-coupled RNA processing events. One important mark is phosphorylation at Ser5 position, whose level peaks during transcription initiation but gradually diminishes toward the 3' end of genes. Ssu72 is a known CTD pSer5 phosphatase. Recent studies identified a binding partner of Ssu72, symplekin, which is an essential scaffold protein in pre-mRNA 3' end processing. Little is known about the molecular function of symplekin and neither do we understand how the symplekin-Ssu72 interaction couples pre-mRNA 3' processing to transcription. We first determined the crystal structure of the symplekin-Ssu72-CTD phosphopeptide complex. The N-terminal domain of symplekin embraces Ssu72 with its HEAT-repeat motif, serving as a typical molecular scaffold.

Strikingly, the CTD phosphopeptide bound to the active site of Ssu72 has the peptide bond between pSer5 and Pro6 in the cis configuration, distinct from all known CTD conformations, which were exclusively in trans. While it was generally believed that only the trans peptide bond is recognized by proline-directed serine/threonine phosphatases or kinases, our discovery demonstrates for the first time that Ssu72 targets the energetically less-favorable cis peptide bond. In addition, we found that the binding of symplekin and also the presence of a proline cis-trans isomerase can stimulate the phosphatase activity of Ssu72 in vitro. The symplekin-Ssu72 interaction as well as the catalytic activity of Ssu72 is required in our transcription-coupled polyadenylation assay. Overall, our study has important implications for the regulation of RNAP II transcription by cis-trans isomerization of the CTD and will help us understand how CTD modifications influence the recruitment of pre-mRNA 3' end processing factors in a transcription-coupled manner.

Recent studies showed that Ssu72 is also a phosphatase of CTD pSer7, which is involved in small nuclear RNA transcription and 3' end processing. However, a pSer7 phosphatase activity appears to be inconsistent with our structure because pSer7 is followed by Tyr1' of the next repeat rather than a proline, and it is unlikely for the pSer7-Tyr1' peptide bond to be in cis configuration. To solve this conundrum, we determined the crystal structure of the pSer7 CTD peptide bound to Ssu72. Surprisingly, the backbone of the pSer7 CTD runs in an opposite direction compared with the pSer5 CTD, allowing a trans pSer7-Pro6 peptide bond to be accommodated in the active site. However, Ssu72 has a much lower affinity for pSer7 than pSer5 and several structural features are detrimental for the catalytic activity towards pSer7. Consistent with these observations, our in vitro assays showed that the dephosphorylation of pSer7 by Ssu72 is ~4000-fold lower than that of pSer5. This further characterization of Ssu72 not only presents the first phosphatase in the literature that recognizes peptide substrates in both directions but also provides a more comprehensive understanding on CTD regulation by phosphatases from a structural perspective.

Another protein, Rtr1, was recently suggested to function as a pSer5 phosphatase in a zinc-dependent fashion, separately or redundantly with Ssu72. We solved the crystal structure of Rtr1 and discovered a new type of zinc finger with no close structural homologs. Unexpectedly, Rtr1 does not present any evidence of an active site and it lacks detectable phosphatase activity in all our assays. We believe that, based on our results, Rtr1 does not have catalytic ability but instead indirectly regulate the phosphorylation state of the CTD. In summary, our studies on the symplein-Ssu72-CTD complex as well as Rtr1 have revealed several novel structural features that are essential for the CTD regulation at the atomic level. These results will also shed light on understanding the mechanism by which RNAP II transcription and RNA processing are coupled.