Symplekin (Pta1 in yeast) is a scaffold in the large protein complex that is required for 3′-end cleavage and polyadenylation of eukaryotic messenger RNA precursors (pre-mRNAs) 1–4, and also participates in transcription initiation and termination by RNA polymerase II (Pol II) 5,6. Symplekin mediates interactions among many different proteins in this machinery 1,2,7–9, although the molecular basis for its function is not known. Here we report the crystal structure at 2.4 Å resolution of the N-terminal domain (residues 30–340) of human symplekin (Symp-N) in a ternary complex with the Pol II C-terminal domain (CTD) Ser5 phosphatase Ssu72 7,10–17 and a CTD Ser5 phosphopeptide. The N-terminal domain of symplekin has the ARM or HEAT fold, with seven pairs of anti-parallel α-helices arranged in the shape of an arc. The structure of Ssu72 has some similarity to that of low-molecular-weight phosphotyrosine protein phosphatase 18,19, although Ssu72 has a unique active site landscape as well as extra structural features at the C-terminus that is important for interaction with symplekin. Ssu72 is bound to the concave face of symplekin, and engineered mutations in this interface can abolish interactions between the two proteins. The CTD peptide is bound in the active site of Ssu72, unexpectedly with the pSer5-Pro6 peptide bond in the cis configuration, which contrasts with all other known CTD peptide conformations 20,21. While the active site of Ssu72 is about 25 Å away from the interface with symplekin, we found that the symplekin N-terminal domain stimulates Ssu72 CTD phosphatase activity in vitro. Furthermore, the N-terminal domain of symplekin inhibits polyadenylation in vitro, but importantly only when coupled to transcription. As catalytically active Ssu72 overcomes this inhibition, our results demonstrate a role for mammalian Ssu72 in transcription-coupled pre-mRNA 3′-end processing.

SUMMARY We recently reported that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5′-end capped mRNAs. Here we report that their mammalian homolog, Dom3Z, possesses pyrophosphohydrolase, decapping and 5′-3′ exoribonuclease activities, and will be referred to as DXO. Surprisingly, we find that DXO preferentially degrades defectively capped pre-mRNAs in cells. Further studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, in contrast to current understanding, and poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at up to 1.5Å resolution provide elegant insights into the catalytic mechanism and molecular basis for its three apparently distinct activities. Our data reveal a pre-mRNA 5′-end capping quality control mechanism in mammalian cells, with DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5′-end capping and subsequent pre-mRNA processing.

A recently developed human norovirus cell culture system revealed that the presence of bile enhanced or was an essential requirement for the growth of certain genotypes. Before this discovery, histo-blood group antigens (HBGAs) were the only well-studied cofactor known for human noroviruses, and there was evidence that several genotypes poorly bound HBGAs. Therefore, the purpose of this study was to investigate how human norovirus capsids interact with bile acids. We found that bile acids had low-micromolar affinities for GII.1, GII.10, and GII.19 capsids but did not bind GI.1, GII.3, GII.4, or GII.17. We showed that bile acid bound at a partially conserved pocket on the norovirus capsid-protruding (P) domain using X-ray crystallography. Amino acid sequence alignment and structural analysis delivered an explanation of selective bile acid binding. Intriguingly, we discovered that binding of the bile acid was the critical step to stabilize several P domain loops that optimally placed an essential amino acid side chain (Asp375) to bind HBGAs in an otherwise HBGA nonbinder (GII.1). Furthermore, bile acid enhanced HBGA binding for a known HBGA binder (GII.10). Altogether, these new data suggest that bile acid functions as a loop-stabilizing regulator and enhancer of HBGA binding for certain norovirus genotypes. IMPORTANCE Given that human norovirus virions likely interact with bile acid during a natural infection, our evidence that an HBGA nonbinder (GII.1) can be converted to an HBGA binder after bile acid binding is of major significance. Our data provide direct evidence that, like HBGAs, bile acid interaction on the capsid is an important cofactor for certain genotypes. However, more unanswered questions seem to arise from these new discoveries. For example, is there an association between the bile acid requirement and the prevalence of certain genotypes? That is, the GII.1 and GII.10 (bile acid binders) genotypes rarely caused outbreaks, whereas the GII.4 and GII.17 genotypes (bile acid nonbinders) were responsible for large epidemics. Therefore, it seems plausible that certain genotypes require bile acids, whereas others have modified their bile acid requirements on the capsid.

Human noroviruses are the leading cause of acute gastroenteritis in humans. Noroviruses also infect animals, such as cows, mice, cats, and dogs. How noroviruses bind and enter host cells is still incompletely understood. Recently, the type I transmembrane protein CD300lf was identified as the murine norovirus receptor, yet it is unclear how the virus capsid and receptor interact at the molecular level. In this study, we determined the X-ray crystal structure of the soluble CD300lf (sCD300lf) and the murine norovirus capsid protruding domain complex at a 2.05-Å resolution. We found that the sCD300lf-binding site is located on the topside of the protruding domain and involves a network of hydrophilic and hydrophobic interactions. sCD300lf locked nicely into a complementary cavity on the protruding domain that is additionally coordinated with a positive surface charge on sCD300lf and a negative surface charge on the protruding domain. Five of six protruding domain residues interacting with sCD300lf were maintained between different murine norovirus strains, suggesting that sCD300lf was capable of binding to a highly conserved pocket. Moreover, a sequence alignment with other CD300 paralogs showed that the sCD300lf-interacting residues were partially conserved in CD300ld but variable in other CD300 family members, consistent with previously reported infection selectivity. Overall, these data provide insights into how a norovirus engages a protein receptor and will be important for a better understanding of selective recognition and norovirus attachment and entry mechanisms. Noroviruses exhibit exquisite host range specificity due to species-specific interactions between the norovirus capsid protein and host molecules. Given this strict host range restriction, it has been unclear how the viruses are maintained within a species between relatively sporadic epidemics. While much data demonstrate that noroviruses can interact with carbohydrates, recent work has shown that expression of the protein CD300lf is both necessary and sufficient for murine norovirus infection of mice and binding of the virus to permissive cells. Importantly, the expression of this murine protein by human cells renders them fully permissive for murine norovirus infection, indicating that at least in this case, host range restriction is determined by molecular events that control receptor binding and entry. Defining the atomic-resolution interactions between the norovirus capsid protein and its cognate receptor is essential for a molecular understanding of host-range restriction and norovirus tropism.