Molecular mechanisms for co-transcriptional assembly of ribonucleoproteins

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

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Abstract

We are broadly interested in how ribonucleoproteins (RNPs) 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. We are currently generating data on in vitro reconstituted and cell extract-based systems to study three areas of RNP biology: spliceosome assembly, ribosome assembly, and mRNA regulation by small RNAs during transcription. Research has generally focused on the question of how proteins and RNAs find target sites in nascent RNA to assemble an RNP important for gene expression. Many RNPs target 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. Using a single-molecule imaging approach, we can define the kinetic mechanisms driving the target search process in real time and unravel the molecular details of the assembly pathway. This approach also allows us to uncover rare and short-lived intermediates that are critical to the assembly pathway. In this fiscal year, we have written a review article published in the Journal of Molecular Biology that highlights recent advances in single-molecule approaches to study RNP assembly mechanisms and identifies open research questions in the field. One project in the lab focuses on identifying how regulation is coordinated when multiple RNAs target the same site. Bacteria often must respond to multiple stress conditions at the same time by employing multiple small RNAs to alter gene expression. Using single-molecule methods, we have identified divergent kinetic mechanisms utilized by multiple small RNAs to regulate the same mRNA despite having similar base-pairing potential with the target site. This suggests that there may be programmed kinetic selection of sRNA regulation when a bacterial encounters multi-stress conditions. These results are in progress towards publication. For this project, we have also established a collaboration with Giesla Storz's lab (NICHD) at the NIH to examine the process of co-transcriptional small RNA targeting in vivo using a reporter system. Additionally, we have begun to collaborate with Susan Gottesman's lab (NCI) to understand the multiple sRNA regulators of a particular mRNA (the rpoS mRNA), which governs the bacterial general stress response. In parallel projects, we are leveraging single-molecule approaches to study the mechanisms of eukaryotic ribosome assembly and spliceosome assembly. Using a cell extract-based system, we have reconstituted the first step of eukaryotic ribosome assembly - association of the seven protein-containing UtpA complex with precursor ribosomal RNA (pre-rRNA). We find that UtpA associates remarkably stably with pre-rRNAs as short as 90 nucleotides supporting previous crosslinking, immunoprecipitation, and Cryo-EM studies that indicate UtpA binds close to the 5' end of the RNA. These studies lay the foundation for characterizing the next steps of assembly: association of the UtpB and the U3 small nucleolar RNP. This ongoing study represent the first in vitro reconstitution of de novo eukaryotic ribosome assembly underscoring the impact on the field. We have also recapitulated the first step of co-transcriptional splicing by examining recruitment of the U1 small nuclear RNP (snRNP) to purified RNA polymerase II. This ongoing work aims to understand how the 5' splice site is rapidly recognized by the U1 small nuclear RNP during co-transcriptional splicing.