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Transcript-specific determinants of pre-mRNA splicing revealed through in vivo kinetic analyses of the 1st and 2nd chemical steps

ABSTRACT: Understanding how the spliceosome processes its composite of substrates through the two chemical steps required for mRNA production will be essential to deciphering splicing regulation and mis-regulation. Here we measure the in vivo rates of these steps across the genome by coupling metabolic RNA labeling, targeted sequencing, and first order kinetic modeling. We reveal a wide variety of rates by which introns are removed, that splice site sequences are primary determinants of first step rates, and that the second step is generally faster than the first. We show that ribosomal protein genes (RPGs) are spliced faster than non-RPGs at each step, and that RPGs share evolutionarily conserved cis-features which facilitate their splicing. A genetic variant defective at the first step shows the expected defect in the first step, but an unexpected change in the second step which suggests how co-transcriptional splicing functions as an important determinant of splicing rates.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE159665 | GEO | 2021/03/12

REPOSITORIES: GEO

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Project description:Memory B cells play essential roles in the maintenance of long-term immunity and may be important in the pathogenesis of autoimmune disease, but how these cells are distinguished from their naÃ¯ve precursors is poorly understood. To address this, it would be important to understand how gene expression differs between memory and naive B cells in order to elucidate memory-specific functions. Using model systems that help overcome the lack of murine memory-specific markers and the low frequency of antigen-specific memory and naÃ¯ve cells, we undertook a global comparison of gene expression between memory B cells and their naive precursors. 1st generation screen:; These data represent our first generation comparison of gene expression between murine naÃ¯ve and memory splenic B cells. NaÃ¯ve NP-binding splenic B cells were FACS purified from unimmunized mVh186.2 transgenic Balb/c mice. Memory B cells were generated by immunizing mVh186.2 transgenic Balb/c mice with 2 doses of NP-CGG in alum delivered i.p. 6 weeks apart. After a minimum of 12-weeks rest, NP-binding splenic B cells were isolated by FACS. Total RNA was extracted, cRNA synthesized and labeled and hybridized to Affymetrix mouse 430 2.0 chips. 2nd generation screen:; These data represent our second generation comparison of gene expression between murine naÃ¯ve and memory splenic B cells. NaÃ¯ve NP-binding AA4.1neg splenic B cells were FACS purified from unimmunized mVh186.2 transgenic Jk KO Balb/c mice. Memory B cells were generated from these naive precursors after adoptive transfer into recipients that mount poor endogenous NP-responses. 12-weeks post i.p. immunization with NP-CGG in alum, NP-specific splenic memory B cells were isolated by FACS. Total RNA was extracted, cRNA synthesized and labeled and hybridized to Affymetrix mouse 430 2.0 chips. Memory/NaÃ¯ve comparison data linked below as Supplementary files. SERIES_CITATION: Tomayko, MM, Anderson, SM, Brayton CE, Sadanand, S, Steinel NC, Behrens, TW, Shlomchik, MJ. 2008. Systematic comparison of gene expression between murine memory and naÃ¯ve B cells demonstrates that memory B cells have unique signaling capabilities. J. Immunol., in press, 2008. Experiment Overall Design: 1st generation screen: 7 samples, 3-4 biological replicates each of murine splenic naÃ¯ve and memory B cells Experiment Overall Design: 2nd generation screen: 8 samples, 4 biological replicates each of murine splenic naÃ¯ve and memory B cells

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Transcript-specific determinants of pre-mRNA splicing revealed through in vivo kinetic analyses of the 1st and 2nd chemical steps

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