Mark T. McNally, PhD
Microbiology and Molecular Genetics
Medical College of Wisconsin
Research Focus: RNA Processing (splicing and polyadenylation) in Cells and Viruses
PhD: SUNY Buffalo (1988) Molecular Biology
My laboratory uses molecular, genetic, and biochemical approaches to study post-transcriptional mechanisms of gene regulation, including RNA splicing and polyadenylation control. The primary model system used is the simpler retrovirus, Rous sarcoma virus (RSV), although the lab also studies regulation of cellular genes.
RNA Splicing Control
Recent advances from genomic, bioinformatic, and RNA splicing array approaches have demonstrated that alternative splicing and alternative polyadenylation are very widespread in humans, and deregulation of alternative splicing (and to a lesser extent, polyadenylation) is commonly responsible for human disease. It is also now appreciated that many RNA processing events, including splicing and polyadenylation, are coupled. Thus, there is great interest in understanding the mechanisms of alternative splicing/polyadenylation and how coupling occurs. Retroviruses such as RSV exploit the host cell RNA processing machinery in interesting ways and serve as model systems to study RNA processing. RSV generates subgenomic (spliced) mRNAs, but the majority of the primary transcripts remains unspliced and serves as mRNA for structural proteins and as genome for progeny virions. In contrast, host RNAs are usually spliced to completion, so the virus has evolved mechanism to repress splicing of viral RNA. The accumulation of RSV unspliced RNA is in part explained by the action of a novel cis-acting RNA element, the negative regulator of splicing, or NRS, which acts as a pseudo 5' ss that serves to repress splicing at the RSV alternative 3' splice sites. The viral element can also repress splicing of heterologous pre-mRNAs in cells and model pre-mRNAs in vitro. Two separate sequences within the minimal ~227 nt NRS are required for inhibition: a purine-rich 5' sequence and a distant 3' region that contains splice site-like sequences. The purine-rich region binds several proteins of the "SR protein" family, which are proteins that play critical roles in several steps in RNA splicing. The 3' region binds two small nuclear rib nucleoprotein complexes (snRNPs) required for conventional mRNA splicing, U1 and U2, and an additional snRNP (U11) that replaces U1 in a minor spliceosome that removes a rare class of introns. The SR proteins are required for U1 binding, which correlates with U1 binding. Since the U1 and U11 sites overlap, U11 may modulate the inhibitory effect of U1 binding. NRS can then interact with the viral splice sites, but an aberrant non-productive NRS splicing complex is formed that sequesters the viral 3'ss and prevents their association with the authentic 5'ss; splicing inhibition results (see Figure below). Elucidation of why splicing does not occur from the NRS and the molecular details of splicing inhibition continue to be a major focus of the lab.
Because the processes of RNA splicing and polyadenylation are coupled, the means by which unspliced viral RNA is efficiently polyadenylated is also of interest. Interestingly, NRS deletions not only cause over splicing of viral RNAs but also increased read through past the viral polyA site, indicating that the NRS also promotes polyadenylation. We showed that mutations that affect splicing control simultaneously cause decreased 3' end formation. Our recent studies showed that the viral polyadenylation site is inherently weak due to suboptimal auxiliary poly(A) signals and poor binding of the polyadenylation factor CstF. We also made the novel finding that SR proteins, in the context of the inhibitory NRS complex, stimulate use of the weak RSV poly(A) site. Work continues in the lab to understand this novel link between the splicing and polyadenylation machineries.
hnRNP H Family Members in Viral and Cellular splicing Control
U11 is a low-abundance snRNP that replaces U1 in a minor-class spliceosome that excises a rare class of non-canonical introns. While U11 snRNP does not contribute to NRS splicing inhibition or poly(A) promotion, it binds the NRS quite well and could 'fine tune' the system by blocking U1 binding to an overlapping binding site. The NRS binds U11 about as well as U1, despite it being ~100-fold less abundant. We've shown that U11 can bind the NRS as a mono-snRNP (in contrast to authentic splicing substrates) and that a G-rich element just downstream of the U11 consensus is required for efficient binding. The G-rich element binds hnRNP H, and hnRNP H is required for optimal U11 binding to the NRS in vitro and in vivo. Interestingly, ~20% of authentic minor-class introns harbor G-rich sequences in positions analogous to the NRS, and we've shown that splicing of the SCN4A and P120 minor-class introns requires hnRNP H. hnRNP H and a closely-related family member, hnRNP F, also modulate alternative splicing of major-class introns. Curiously, hnRNP F does not appear to function in the minor pathway. Our lab continues to investigate the mechanisms of action of hnRNP H/F in minor- and major-class alternative splicing.
Model for NRS function. A) The NRS. SR proteins bind to the purine-rich region and facilitate binding of U1 snRNP to the 3' end of the NRS. U11 binding does not require the high-affinity SR sites; hnRNP H promotes binding of U11. A similar mechanism promotes splicing of some cellular minor-class introns. B) U1 snRNP bound to the NRS, which is in a proximal position relative to the authentic 5'ss, interacts with factors associated with the 3' ss, similarly to the way normal intron bridging interactions occur. U1 is thought to be replaced by U6 snRNP and a spliceosomal complex is formed (U4 and U5 omitted for clarity), but splicing does not occur from the NRS (red X); the nonproductive splicing complex sequesters the 3' ss and prevents its pairing with the authentic 5'ss, resulting in splicing inhibition. U11 binding would prevent U1 binding and assembly of the NRS complex, and allow the potential for viral RNA splicing. The NRS complex also promotes polyadenylation of unspliced viral RNAs. Within the NRS complex, SR proteins simulate polyadenylation; the mechanism that is under investigation.