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Zappulla, D. C.

Publications and source records attributed to Zappulla, D. C..

2 recordsLinked to original sources
bioRxiv2017

Repositioning the Sm-binding site in S. cerevisiae telomerase RNA reveals RNP organizational flexibility and Sm-directed 3′-end formation

Telomerase RNA contains a template for synthesizing telomeric DNA by reverse transcription and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase subunits Est1 and Ku bind to the telomerase RNA, TLC1, and it has been shown that these proteins still function when their binding sites are repositioned within the RNA. TLC1 is also bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)-) TLC1 isoform. Here, we first show that Sm7 can perform this function even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we tested the hypothesis that the location of the Sm7-binding site relative to the 3' end is contrastingly important. When we moved the Sm site to locations 5' of its native position, we observed that this stabilized shorter forms of poly(A)- TLC1 in a manner precisely corresponding to how far upstream the Sm site was moved. This provides strong evidence that the location of Sm7 binding to TLC1 controls where the mature poly(A)- 3' end is formed. In summary, our results show that Sm7 and the 3' end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.

molecular biology↗
bioRxiv2017

A CRISPR-Based Yeast Two-Hybrid System For Investigating RNA-Protein Interactions

Despite the great importance of RNA-protein interactions in cells, there is a very limited set of approaches available for identifying proteins that bind to a specific RNA. We report here combining the use of CRISPR technology with the yeast two-hybrid protein-protein interaction system in order to create an advantageous method for investigating RNA-protein interactions. In this CRISPR-assisted RNA/RBP yeast (CARRY) two-hybrid system, an RNA of interest is targeted to the promoters of standard yeast two-hybrid reporter genes by fusing it to the CRISPR guide RNA in a strain expressing catalytically deactivated Cas9 (dCas9). If the promoter-tethered RNA binds to a protein fused to Gal4 transcriptional activation domain (GAD), then the reporter genes become transcribed, just as in the standard protein-protein yeast two-hybrid assay. We used the CARRY two-hybrid system to analyze MS2 bacteriophage RNA hairpin binding to the MS2 coat protein (MCP). We tested MS2 hairpin mutants with a range of biochemically determined binding affinities for MCP and found that CARRY two-hybrid detected all binding interactions with dissociation constants [≤]300 nM. In summary, this new CRISPR-based yeast two-hybrid system provides an easily operable, much-needed new tool for identifying proteins that bind to a particular RNA.

molecular biology↗