We study the mechanisms of two essential nanomachines of gene expression, RNA polymerase II (Pol II) and the spliceosome, using brewer’s yeast as a model system.
Our work on Pol II focuses on transcription termination as a strategy for gene regulation. In the 1990’s we discovered a pathway that uses the helicase Sen1 and a collection of RNA-binding proteins (including Nrd1 and Nab3) to terminate Pol II transcription within ~500 base pairs from the transcription start site (see figure). This “NNS” pathway produces short non-coding RNAs, such as snRNAs and snoRNAs, and also regulates mRNA synthesis by attenuation. We use genetics, genomics, cell biology and biochemistry to define the mechanism and targets of the Sen1-dependent termination pathway. Currently we are investigating the function of the poorly studied RNA-binding protein Hrp1 in the NNS pathway and trying to discern how mutations in the Rpb11 and Rpb3 subunits of Pol II result in defects in NNS terminator recognition. Our goal is to develop a structural model of the Pol II NNS termination complex.
When elongating Pol II (gray) produces a transcript (red) that contains NNS terminator sequences, binding of these sequences by the Nrd1-Nab3-Sen1 proteins results in termination of transcription. Mutations in these proteins, and in Hrp1 and the Rpb11 subunit of Pol II, can cause transcription to continue through certain NNS terminators. Mutations in the Rpb1-CTD phosphatase Ssu72 can also influence recognition of NNS terminators.
Our studies on the spliceosome focus on U6 snRNA and the dynamic RNA-RNA and RNA-protein interactions required for its pairing with U2 snRNA and the intron during activation of the spliceosome for intron excision. U2 and U6 snRNAs make up the catalytic core of the spliceosome and align the intron sequences for the first step of splicing. Before that can happen, U4 snRNA must be unwound from U6 by the helicase Brr2, which is a component of the U5 snRNP. Another U5 snRNP protein, Prp8, regulates this process. We discovered a mutation in U4 snRNA, U4-cs1, that arrests U4/U6 unwinding at low temperature. Isolation of genetic suppressors of U4-cs1 identified amino acid residues in five proteins, including Prp8 and Brr2, that regulate this process (see figure). We are currently using biochemical and genetic methods to better understand the mechanism by which these proteins and RNAs transmit allosteric signals more than 70 Ångstroms across the spliceosome to enable the large conformational changes required for spliceosome activation.