David Alan Brow
I have a long-standing interest in the roles of protein/DNA, protein/RNA and RNA/RNA interactions in eukaryotic gene expression. Current work in my lab focuses on two topics: 1) the mechanism of transcription of the U6 spliceosomal RNA gene by RNA polymerase III, and 2) the role of the U4/U6 small nuclear RNP in pre-mRNA splicing. We use the yeast Saccharomyces cerevisiae as a model organism.
In vivo assembly of an RNA polymerase III transcription complex on a yeast U6 RNA gene.
The yeast U6 RNA gene (SNR6) has a unique promoter structure, which we have exploited to examine the in vivo mechanism of initiation by RNA polymerase III (pol III). Few in vivo studies of pol III transcription complex assembly have been attempted, because of the difficulties posed by the intragenic location of the promoter elements and by the multiple genomic copies of the transcription units being examined. The SNR6 promoter, however, is largely extragenic; mutations in these extragenic elements affect only transcription efficiency and not RNA stability or function. Furthermore, SNR6 is single-copy and essential. Consequently, the growth phenotype of snr6 strains bearing extragenic promoter mutations directly reflects promoter function, allowing straightforward structure/function analyses of the promoter elements.
We have used a combination of mutational analyses and chromatin footprinting studies to define the promoter requirements for in vivo transcription of SNR6 in greater depth than has been accomplished for any other RNA polymerase III transcription unit. Intriguingly, the in vivo promoter requirements are strikingly different than the promoter requirements for transcription in vitro with purified factors and polymerase. With purified components, only upstream sequences are required for transcription, while in vivo, intragenic and downstream elements are required and the upstream elements are nonessential. This dichotomy stems from the binding properties of TFIIIB, the transcription factor that recruits pol III to the promoter. In vitro, purified TFIIIB binds directly to the upstream sequences, via sequence-specific DNA contacts. In vivo, however, binding of TFIIIB is directed primarily by protein:protein interactions with another factor, TFIIIC, which binds to the intragenic and downstream promoter elements. We are using a genetic strategy to identify amino acid/base pair interactions crucial for TFIIIC binding to SNR6 in vivo. We have identified five recessive lethal point mutations in the downstream B block, the primary binding site of TFIIIC. To identify the amino acids in TFIIIC which contact these residues, we are selecting extragenic suppressors of the lethal B block mutations. Since there are no clear DNA binding motifs in the subunit of TFIIIC that binds the B block, the results of this analysis may identify a novel mechanism for sequence-specific recognition of DNA by a protein. The nature of the TFIIIC/B block interaction is of particular interest because the B block promoter element appears to have been recruited from an RNA sequence, the GTYC loop of tRNA.
A consensus TATA box upstream of the U6 RNA coding region is essential for transcription of SNR6 with purified TFIIIB and polymerase, presumably because it is recognized by the TATA-binding protein, which is a subunit of TFIIIB. Surprisingly, a 42 base pair deletion (D42) between the U6 RNA coding region and the downstream B block makes in vivo transcription of SNR6 also highly dependent on the TATA box, presumably by interfering with the function of TFIIIC. We propose that the D42 mutation acts by disrupting the binding of proteins that enhance the function of TFIIIC in vivo.
We are currently examining the chromatin structure of the 250 bp stretch of DNA that separates the upstream TATA box from the downstream B block. Our hypothesis is that a positioned nucleosome or other complex of proteins binds to this region to direct transcription complex assembly. Ultimately, we hope to elaborate the components of the in vivo SNR6 transcription initiation complex, and to define their contribution to its assembly and maintenance.
Dynamics of U6 RNA function in nuclear pre-mRNA splicing
U6 RNA is an essential component of the spliceosome, and may act as a ribozyme to catalyze intron removal. Key to elucidating the role of U6 RNA in pre-mRNA splicing is an understanding of its interaction with U4 RNA. Association of U4 and U6 RNAs, via extensive intermolecular base-pairing, appears to be required for entry of U6 RNA into the spliceosome. However, before the catalytic steps of splicing occur the U4/U6 base-pairing is disrupted so that U6 RNA can interact with other spliceosomal components. We and others have proposed that U4 RNA serves two roles: as a carrier to transport U6 RNA to the spliceosome, and as an anti-sense regulator to suppress U6 RNA function until the spliceosome is properly assembled on an authentic intron. Thus, the U4/U6 interaction is likely pivotal to the mechanism of splicing. Work in my lab has defined a large conformational switch that is required of U6 RNA as it transits the splicing cycle: a stable intramolecular stem must be disrupted to form the U4/U6 base-pairing interaction. Conformational switches in RNA have been intensively sought, so the dynamics of U6 RNA are of interest both for what they will tell us about the mechanism of splicing and as a paradigm for the biochemical activity of RNA in general. We have initiated a genetic analysis of the U6 RNA conformational switch by introducing mutations that hyperstabilize either U6 RNA or the U4/U6 complex. These mutations result in a cold-sensitive (cs) growth defect and the inhibition of spliceosome assembly or function. We have selected over one hundred independent suppressors of the cs growth defect caused by the mutations in U6 and U4 RNAs. About one third of these are cis-suppressors, i.e., additional mutations in the U6 or U4 RNA genes. Analysis of the cis-suppressors confirms the secondary structure models of U6 RNA and the U4/U6 complex, and identifies regions in the RNAs that influence spliceosome assembly. Initial characterization of the trans-suppressors has identified mutations in the wild type partner RNA (U4 or U6), in two known splicing factors, and in at least one as yet unidentified gene. Further characterization of the trans-suppressors is expected to elucidate the function of known splicing factors and to reveal previously unknown splicing factors involved in U4/U6 snRNP assembly.
We are also pursuing a biochemical analysis of U6 and U4 RNAs from both yeast and humans. Using thermal denaturation studies, we are measuring the relative stabilities of secondary structure elements in wild type and mutant yeast U6 RNA. This will allow us to correlate the structural and physiological effects of mutations in U6 RNA. In the course of this work we identified a structural element in human U6 RNA that destabilizes the deproteinized U4/U6 RNA complex in vitro. This element may play a central role in dissociation of the U4/U6 snRNP during activation of the spliceosome.