Nucleotide sequences of the 5' termini of phi X174 mRNAs synthesized in vitro

When using X174 RFI DNA as a template, in vitro, E. coli RNA polymerase synthesizes four major purine triphosphate-containing 5′ end sequences. RNase A digests of α³²P labeled RNA were further digested with spleen exonuclease to remove the bulk of the oligonucleotides with 5′ hydroxyls and then chromatographed on DEAE cellulose to resolve the remaining 5′ terminal oligonucleotides. By application of standard separation and sequence techniques, the major 5′ end sequences were shown to be: pppApUp(Cp), pppApApApUp(Cp), pppApApApApUp(Cp), and pppGpApUp(Gp).     

Histone Deacetylases and Phosphorylated Polymerase II C-Terminal Domain Recruit Spt6 for Cotranscriptional Histone Reassembly

Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C terminus that recognizes Pol II C-terminal domain (CTD) peptides phosphorylated on Ser2, Ser5, or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3′ ends of genes, where phosphorylated Ser2 reaches its maximum level. In addition, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3′ ends of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation, and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo.     

Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5′ cyclization sequence (5′CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5′CS, and the presence of DCS-PK facilitates the formation of 5′-3′ RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.     

Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats

Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5′-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5′-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5′-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5′-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5′-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.     

Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA

The mRNA export complex TREX (TREX) is known to contain Aly, UAP56, Tex1 and the THO complex, among which UAP56 is required for TREX assembly. Here, we systematically investigated the role of each human TREX component in TREX assembly and its association with the mRNA. We found that Tex1 is essentially a subunit of the THO complex. Aly, THO and UAP56 are all required for assembly of TREX, in which Aly directly interacts with THO subunits Thoc2 and Thoc5. Both Aly and THO function in linking UAP56 to the cap-binding protein CBP80. Interestingly, association of UAP56 with the spliced mRNA, but not with the pre-mRNA, requires Aly and THO. Unexpectedly, we found that Aly and THO require each other to associate with the spliced mRNA. Consistent with these biochemical results, similar to Aly and UAP56, THO plays critical roles in mRNA export. Together, we propose that Aly, THO and UAP56 form a highly integrated unit to associate with the spliced mRNA and function in mRNA export.     

Human mRNA export machinery recruited to the 5' end of mRNA

Pre-mRNAs undergo splicing to remove introns, and the spliced mRNA is exported to the cytoplasm for translation. Here we investigated the mechanism for recruitment of the conserved mRNA export machinery (TREX complex) to mRNA. We show that the human TREX complex is recruited to a region near the 5' end of mRNA, with the TREX component Aly bound closest to the 5' cap. Both TREX recruitment and mRNA export require the cap, and these roles for the cap are splicing dependent. CBP80, which is bound to the cap, associates efficiently with TREX, and Aly mediates this interaction. Together, these data indicate that the CBP80-Aly interaction results in recruitment of TREX to the 5' end of mRNA, where it functions in mRNA export. As a consequence, the mRNA would be exported in a 5' to 3' direction through the nuclear pore, as observed in early electron micrographs of giant Balbiani ring mRNPs.     

Chtop is a component of the dynamic TREX mRNA export complex

The TREX complex couples nuclear pre‐mRNA processing with mRNA export and contains multiple protein components, including Uap56, Alyref, Cip29 and the multi‐subunit THO complex. Here, we have identified Chtop as a novel TREX component. We show that both Chtop and Alyref activate the ATPase and RNA helicase activities of Uap56 and that Uap56 functions to recruit both Alyref and Chtop onto mRNA. As observed with the THO complex subunit Thoc5, Chtop binds to the NTF2‐like domain of Nxf1, and this interaction requires arginine methylation of Chtop. Using RNAi, we show that co‐knockdown of Alyref and Chtop results in a potent mRNA export block. Chtop binds to Uap56 in a mutually exclusive manner with Alyref, and Chtop binds to Nxf1 in a mutually exclusive manner with Thoc5. However, Chtop, Thoc5 and Nxf1 exist in a single complex in vivo. Together, our data indicate that TREX and Nxf1 undergo dynamic remodelling, driven by the ATPase cycle of Uap56 and post‐translational modifications of Chtop.