Multi-step recognition of potential 5' splice sites by the Saccharomyces cerevisiae U1 snRNP
- University of Wisconsin-Madison, United States
- New England Biolabs, United States
Abstract
In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5' splice site (5' SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5' SS recognition have largely been done in vivo or in cellular extracts where the U1/5' SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5' SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5' SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS and preferentially accumulate at these sequences rather than on close cognates.
Data availability
Source data files have been provided for Figure 1-Supplemental Figure 2. Due its large size, the source data for the single molecule microscopy experiments will be hosted by a campus web server and freely available for public download using Globus at the weblink belowhttps://app.globus.org/file-manager?origin_id=2b62cfc8-0c02-42ca-bb75-1a257d7b4284&origin_path=%2FWe have included this link in the manuscript text with the materials and methods section describing single molecule data collection.
Article and author information
Author details
Funding
National Institutes of Health (R01 GM122735)
- Aaron A Hoskins
National Institutes of Health (R35 GM136261)
- Aaron A Hoskins
National Institutes of Health (R35 GM126914)
- Lloyd M Smith
National Institutes of Health (T32 GM008505)
- Sarah R Hansen
National Institutes of Health (F32 GM143780)
- David S White
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Hansen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,410
- views
-
- 316
- downloads
-
- 6
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Multi-step recognition of potential 5' splice sites by the Saccharomyces cerevisiae U1 snRNP
eLife 11:e70534.
https://doi.org/10.7554/eLife.70534
Further reading
-
- Biochemistry and Chemical Biology
Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small-molecule-induced proteasome substrates.
-
- Biochemistry and Chemical Biology
Several metabolites have been shown to have independent and at times unexpected biological effects outside of their metabolic pathways. These include succinate, lactate, fumarate, and 2-hydroxyglutarate. 2-Hydroxybutyrate (2HB) is a byproduct of endogenous cysteine synthesis, produced during periods of cellular stress. 2HB rises acutely after exercise; it also rises during infection and is also chronically increased in a number of metabolic disorders. We show here that 2HB inhibits branched-chain aminotransferase enzymes, which in turn triggers a SIRT4-dependent shift in the compartmental abundance of protein ADP-ribosylation. The 2HB-induced decrease in nuclear protein ADP-ribosylation leads to a C/EBPβ-mediated transcriptional response in the branched-chain amino acid degradation pathway. This response to 2HB exposure leads to an improved oxidative capacity in vitro. We found that repeated injection with 2HB can replicate the improvement to oxidative capacity that occurs following exercise training. Together, we show that 2-HB regulates fundamental aspects of skeletal muscle metabolism.
