The Functional Role of Splicing Factors in Transcription and Aging

Rachel Stegeman, Purdue University


Proper gene expression of protein coding genes is an integral part of the health and survival of eukaryotes. Transcription and splicing are two coordinated processes which must occur to allow for the proper expression of protein coding genes. These processes are tightly regulated and any misregulation can lead to dysfunctional proteins which can cause functional decline or disease. Splicing occurs via the spliceosome and its associated proteins, splicing factors, with more than 300 total proteins involved in active splicing. When transcribed gene products are produced, this dynamic macromolecular machine splices out introns and ligates exon-exon junctions back together to produce mRNA. Alternative splicing occurs when alternatively used exons can be included or skipped, and exon-exon junctions can be differentially used to produce multiple transcripts. Additionally trans-acting splicing factors bind to RNA elements, and this helps to determine the splicing outcome of a particular transcript, however exactly how splicing factors affect splice site choices from transcript to transcript is still unclear, and is still a highly investigated topic. Our studies will focus on how spliceosomal components and splicing factors play a role in gene regulation, through transcription, or changes in alternative splicing which can cause a functional decline with age. Genome-wide studies of aging have identified subsets of genes that show age-related changes in expression. Aging is associated with a broad induction of stress response pathways. In contrast, a wide variety of functional classes of genes are downregulated with age, often including tissue-specific genes. Whereas the upregulation of age-regulated genes is likely to be governed by stress-responsive transcription factors, questions remain as to why particular genes are susceptible to age-related transcriptional decline. Additionally recent findings showing that splicing is misregulated with age. While defects in splicing could lead to changes in protein isoform levels, they could also impact gene expression through nonsense-mediated decay of intron-retained transcripts. Moreover, the considerable variation between genome-wide aging expression studies indicates that there is a critical need to analyze the transcriptional signatures of aging in single cell types rather than whole tissues. Since age-associated decreases in gene expression could contribute to a progressive decline in cellular function, understanding the mechanisms that determine the aging transcriptome provides a potential target to extend healthy cellular lifespan. Our initial studies demonstrate an example of how splicing and transcription can be linked through a single complex. These processes are coordinated both temporally and spatially, but the regulatory mechanisms governing this coordination are still not fully understood. Here we have identified an interaction between spliceosomal components and the transcriptional machinery which provides an intriguing link between the coupled processes of transcription and splicing. We show that two components of the SF3B complex that forms part of the U2 small nuclear ribonucleoprotein particle (snRNP), SF3B3 and SF3B5, are also subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex in Drosophila melanogaster. Whereas SF3B3 had previously been identified as a human SAGA subunit, SF3B5 had not been identified as a component of SAGA in any species. We also show that SF3B3 and SF3B5 bind to SAGA independent of RNA, and interact with multiple SAGA subunits including Sgf29 and Spt7 in a yeast two-hybrid assay. Through analysis of sf3b5 mutant flies, SF3B5 does not appear to function in SAGA’s histone modifying activities, but is required for expression of a subset of SAGA-regulated genes independent of splicing. Thus, our data support an independent function of SF3B5 in SAGA’s transcription coactivator activity that is separate from its role in splicing. Next, we sought to characterize the contribution of proper splicing to aging using the Drosophila eye as a model system. Several studies have shown that the aging transcriptome is characterized by changes in alternative splicing. However, it is unclear whether these age-related changes in splicing also contribute to the progressive functional decline associated with aging. Since decreased expression of splicing factors is observed in aging photoreceptors, we sought to characterize the contribution of individual age-regulated splicing factors to age-associated changes in alternative splicing and visual function in the Drosophila eye. Here, we show that there are age-related changes in splicing of genes involved in visual function in photoreceptors, and that proper splicing of these genes requires the combinatorial activity of individual age-regulated splicing factors. Notably, all of the splicing factors tested were necessary for proper visual function and/or photoreceptor viability in the adult eye. These data suggest that the coordinated activity of multiple splicing factors is necessary for proper splicing in the eye and to prevent visual senescence. Altogether our studies highlight the importance of splicing factors in gene regulation through transcriptional co-activation and in regulating genes important for cell specific functions during aging.




Weake, Purdue University.

Subject Area

Biochemistry|Molecular biology|Genetics

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