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Shalini Oberdoerffer, Ph.D.
It is presently estimated that greater than 90% of human genes undergo alternative pre-mRNA splicing, and aberrant splicing has been linked to a variety of human pathologies. Alternative splicing decisions are determined by the ability of weak splice sites to effectively compete with strong splice sites for detection by the spliceosome. There is substantial evidence indicating that spliceosome assembly occurs on nascent RNA co-transcriptionally. RNA polymerase II (pol II) carboxy terminal domain (CTD) associated splicing factors detect enhancer or silencer sequences encoded within the newly synthesized transcript to promote or inhibit spliceosome assembly, respectively. Variations in splicing factor expression can thereby result in alternative splicing. Similarly, the rate of pol II transcription elongation can influence alternative splicing, wherein a slow transcription rate favors spliceosome assembly at weak splice sites. A surprising result of recent genome-wide chromatin-immunoprecipation-sequencing (ChIP-seq) studies is the non-random distribution of several epigenetic marks in exons relative to introns. In particular, exons display elevated nucleosome density, DNA methylation and specific histone marks relative to introns. Collectively, these studies raise the possibility that epigenetic modifications may be maintained on DNA to aid the spliceosome in the process of exon definition, and that differential chromatin assembly may represent a critical aspect of alternative splicing regulation.
Processing of CD45 pre-mRNA is a well-established model system to study the regulatory mechanisms of alternative splicing. CD45 is a trans-membrane protein tyrosine phosphatase that initiates signaling through antigen receptors by dephosphorylating the inhibitory tyrosine on Src family kinases. Variable exclusion of exons 4-6 of CD45 transcripts is tightly correlated with stages of lymphocyte development. The goal of my research program is to study RNA and DNA mediated regulatory mechanisms governing differentiation-associated CD45 alternative splicing, with an additional focus on related global shifts in the transcriptome.
RNA-binding splicing factors
Splicesome assembly at weak splice sites is facilitated by the binding of positive acting trans-factors, such as SR proteins, at proximal enhancer sequences. Conversely, the binding of negative acting trans-factors, such as the heterogeneous nuclear ribonucleoproteins (hnRNPs), to silencer sequences can occlude SR protein assembly at enhancers. We previously identified heterogeneous ribonucleoprotein L-like (hnRNPLL) as an activation-induced regulator of the CD45RA to CD45RO transition in peripheral lymphocytes. HnRNPLL binds to exons 4 and 6 of CD45 pre-mRNA, leading to their exclusion from mature transcripts. Only a handful of tissue-specific splicing factors have been identified, and through modulation of hnRNPLL expression, we will assess the global impact of hnRNPLL induction on the lymphocyte transcriptome. HnRNPLL is expressed at the terminal stages of T and B cell development, raising the possibility that hnRNPLL may specifically regulate a subset of transcripts required for mature cell maintenance and/or function. To address these and related questions, mice for conditional hnRNPLL deletion or expression have been generated.
DNA-binding splicing factors
As described above, hnRNPLL influences exclusion of exons 4 and 6 of CD45 transcripts, but does not regulate alternative exon 5. Considering the growing evidence for DNA-mediated regulation of spliceosome assembly, we explored the hypothesis that exon 5 inclusion is modulated by the epigenetic structure of the gene encoding CD45, PTPRC. We found that the zinc-finger DNA-binding protein, CTCF, binds to PTPRC exon 5 and supports inclusion of exon 5 in CD45 transcripts by promoting local pol II pausing. In contrast, de novo methylation of exon 5 DNA during peripheral lymphocyte maturation leads to CTCF eviction and exclusion of exon 5 from CD45 mRNA. Combined genome-wide ChIP-seq and RNA-seq analysis showed that intragenic CTCF functions as a global regulator of alternative splicing. These observations describe a novel category of DNA-binding splicing factors and provide a framework for epigenetic regulation of pre-mRNA processing.
This page was last updated on 6/7/2013.