Molecular mechanisms for co-transcriptional assembly of ribonucleoproteins

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We are broadly interested in how ribonucleoproteins are assembled during transcription of an RNA. The lab was launched in October 2022 and since then we have set up the laboratory for RNA and protein biochemistry, molecular biology, yeast genetics, and single-molecule microscopy. Work has begun to generate in vitro reconstituted and extract-based systems to study three areas of RNP biology: spliceosome assembly, ribosome assembly, and mRNA regulation by small RNAs during transcription. Research has focused on how RNPs find target sequences to base pair with a nascent RNA. Many RNPs function by targeting an RNA through base pairing to specifically guide RNA folding, perform nucleotide modification, or carry out splicing. Targeting often occurs during transcription, such that the target RNA is folding, binding proteins, and being targeted by an RNP at the same time. In work that was published during 2023, we utilized a single-molecule approach to study how a bacterial small RNP, containing a small RNA and the protein Hfq, target a sequence in a nascent mRNA to regulate its expression. This work established a model for efficient recruitment of the small RNP complex to a nascent RNA that involved targeting recently synthesized RNA in proximity to RNA polymerase. In another project completed during the reporting period, we studied how ribosomal proteins facilitate assembly of the bacterial small ribosomal subunit by helping chaperone the ribosomal RNA during transcription. We have begun work to characterize the mechanisms of eukaryotic ribosome assembly and spliceosome assembly. We are particularly interested in the earliest steps of eukaryotic ribosome assembly during transcription. We are also studying association and function of the small nucleolar RNPs which must act on the ribosomal RNA during transcription before the rRNA folds into a stable, native structure. We have also begun studies on how the 5 splice site is recognized by the U1 small nuclear RNP during co-transcriptional splicing. By characterizing the molecular details of RNP assembly, we will provide new understanding of how RNP assembly is dysregulated in disease.