Dual roles of DEAD-box RNA helicase Brr2 in genome stability
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PROVIDER: PRJNA1170866 | ENA |
REPOSITORIES: ENA
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Project description:Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding, a critical step in spliceosomal activation. Brr2 contains an N-terminal domain and two tandem sets of a helicase-like domain followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. The helicase-like domain upstream of Sec63 has clear sequence similarity with domains 1-3 of Hel308. In addition modeling indicates that Brr2 is composed of an N-terminal domain and two consecutive Hel308-like modules (Hel308-I and II). Together this provides our first glimpse of the overall structure of this large and unique spliceosomal ATPase and helicase. Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism to Hel308, that differs from many DEAD-box proteins. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo, potentially serving as a mediator for the regulation of Brr2’s activity by Prp8. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2/Prp8-CTR complex to U4/U6, suggesting a potential role of Prp8-CTR as an auxiliary substrate binding and specificity domain for Brr2.
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Project description:DEAD-box ATPases belong to an abundant class of proteins that are involved in virtually all aspects of RNA metabolism and are found in all kingdoms of life. When bound to a DEAD-box ATPase, the RNA substrate is forced into a kinked conformation that is incompatible with helical structures. Distortion of the RNA can result in unwinding of short RNA duplexes (helicase activity) or destabilize RNA-protein interactions, allowing DEAD-box ATPases to remodel mRNPs (RNPase activity). The RNPase activity makes DEAD-box ATPases suitable molecular building blocks for the implementation of checkpoints that confer directionality to the process of RNA biogenesis. Here, we provide data that characterizes the DEAD-box ATPase Dbp2 (SPBP8B7.16c) of the fission yeast Schizosaccharomyces pombe. Using calibrated RNAPII-ChIP-seq, we determined the transcription profiles of a conditional depletion strain of Dbp2 and the corresponding wild type. For this, we placed the endogenous dbp2 gene under the control of the P.nmt1 promoter, which is repressed in the presence of thiamine. Cells were crosslinked at the beginning (t0) or the end (t9) of a 9h shift to thiamine-containing YES medium. S. cerevisiae spike-in cells were added in a 1:5 OD600 ratio immediately before crosslinking.
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