Biology subjects

Hass, E. P.

Publications and source records attributed to Hass, E. P..

5 recordsLinked to original sources

bioRxiv2022

Massively Parallel Dissection of RNA in RNA-protein interactions in vivo

Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to molecular biology. Here, we present MPRNA-immunoprecipitation (MPRNA-IP), an adaptation of the previously developed massively parallel RNA assay (MPRNA), and a new avenue for in vivo high-throughput dissection of RNA-protein interactions. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, we are able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest including NORAD, MS2, and human telomerase RNA, and we describe statistical models for identifying RNA domains and parsing the structural contributions of RNA in these interactions. By blending modern and classical approaches, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions.

genomics↗

bioRxiv2020

A 4-base pair core-enclosing helix in telomerase RNA is essential and binds to the TERT catalytic protein subunit

The telomerase RNP counters the chromosome end-replication problem, completing genome replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse transcriptase (TERT). Although the majority of the 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). Here, we show that 4-base pairs of core-enclosing helix is required for telomerase to be active in vitro and to maintain yeast telomeres in vivo, whereas {Delta}CEH, 1-bp, and 2-bp alleles do not support telomerase function. Using the CRISPR/dCas9-based "CARRY two-hybrid" assay to assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to TERT, but the shorter-CEH constructs do not, consistent with the telomerase activity and in vivo complementation results. Thus, the CEH is essential in yeast telomerase RNA because it is needed to bind TERT to form the core RNP enzyme. Although the 8 nucleotides that form this 4-bp stem at the base of the CEH are nearly invariant among Saccharomyces species, our results with sequence-randomized and truncated-CEH helices strongly suggest that this binding interaction with TERT is dictated more by secondary than primary structure. In summary, we have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the catalytic core of this biomedically important RNP enzyme.

molecular biology↗

bioRxiv2019

Single-cell imaging reveals unexpected heterogeneity of TERT expression across cancer cell lines

Telomerase is pathologically reactivated in most human cancers, where it maintains chromosomal telomeres and allows immortalization. Because telomerase reverse transcriptase (TERT) is usually the limiting component for telomerase activation, numerous studies have measured TERT mRNA levels in populations of cells or in tissues. However, little is known about TERT expression at the single-cell and single-molecule level. Here we analyzed TERT expression across 10 human cancer lines using single-molecule RNA FISH and made several unexpected findings. First, there was substantial cell-to-cell variation in number of transcription sites and ratio of transcription sites to gene copies. Second, previous classification of lines as having monoallelic or biallelic TERT expression was found to be inadequate for capturing the TERT gene expression patterns. Finally, TERT mRNA had primarily nuclear localization in cancer cells and induced pluripotent stem cells (iPSCs), in stark contrast to the expectation that mature mRNA should be predominantly cytoplasmic. These data reveal unappreciated heterogeneity, complexity, and unconventionality in TERT expression across human cancer cells.

cancer biology↗

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↗