Pre-mRNA processing factors are recruited from nuclear speckles to nascent pre-mRNA transcripts during gene expression. Son may have a dual role in the nucleus to organize pre- mRNA processing machinery in nuclear speckles, and to regulate gene expression. The latter possibility is addressed here and is based on several observations: 1) Son is a large protein with many novel repeat motifs that are not found in any other human protein;these sequences might support a novel role for Son as a scaffolding protein at transcription sites;2) Son localizes at a heterochromatic U2OS 2-6-3 gene locus and is lost upon gene activation;3) several reports show that Son is a DNA binding protein that can influence the efficacy of virus production, both positively and negatively and 4) a genome-wide screen indicates that hundreds of human genes require Son for proper splicing. The experimental focus of this proposal is to use in situ approaches and high-resolution microscopy techniques to study the functions of Son in the context of intact cells. Reported roles for Son in regulation of nuclear speckle organization, replication of hepatitis B virus and influenza virus, in the mitotic spindle apparatus and in anti- apoptotic response gives human disease relevance for studying Son functions.
The specific aims of this project will: 1. Define Son-chromatin interactions. It will be determined if Son is required for maintaining silencing of the U2OS 2-6-3 locus or for recruiting molecular complexes during transcription activation of this locus. The regions of Son that associate with the U2OS 2-6-3 gene locus as well as the regions of the locus where Son associates on the locus will be determined. Son will be depleted by RNAi to examine how chromatin dynamics, histone modifications, chromatin proteins and chromatin remodeling factors are altered at the inactive locus as well as during transcription activation. Endogenous human gene loci that associate with Son will be identified. 2. Determine what alternative splicing events are regulated by Son. We will validate alternative splicing events that require Son by using the most significantly affected pre-mRNA targets already identified by exon arrays. We will define what different types of splicing defects such as exon skipping, alternate 5'splicing, and alternate 3'splicing, are regulated by Son. Inducible minigenes will be constructed for representative Son-dependent exon skipping events, and stable cell lines will be generated to observe alternative splicing in situ. This project will begin to build models for studying Son-dependent splicing in the context of chromatin structure.
The cellular localization of Son in nuclear speckles makes it a likely candidate for gene regulation, exemplified Son repression of the hepatitis B virus core promoter leading to reduced replication (Sun et al., 2001) and Son promotion of influenza virus trafficking and viral RNA production (Karlas et al., 2010). Son is an anti-apoptotic (Sun et al. 2001) mitotic spindle-associated (Nousiainen et al. 2006) protein that is required for cell growth/survival, cell cycle progression and nuclear speckle organization (Sharma et al, 2010;Ahn et al., 2008). The implications for Son in gene regulation, cell death response, association with mitotic spindle and involvement in virus replication underscores the public health relevance for studying Son with regard to its functions in human diseases such as HBV/influenza viral infection and cancer.
|Varia, Sapna; Cheedu, Divya; Markey, Michael et al. (2017) Alignment of Mitotic Chromosomes in Human Cells Involves SR-Like Splicing Factors Btf and TRAP150. Int J Mol Sci 18:|
|Kim, Jung-Hyun; Shinde, Deepali N; Reijnders, Margot R F et al. (2016) De Novo Mutations in SON Disrupt RNA Splicing of Genes Essential for Brain Development and Metabolism, Causing an Intellectual-Disability Syndrome. Am J Hum Genet 99:711-719|
|Battini, Vishnu Priya; Bubulya, Athanasios; Bubulya, Paula A (2015) Accurate splicing of HDAC6 pre-mRNA requires SON. Int J Mol Sci 16:5886-99|
|Lu, Xinyi; Ng, Huck-Hui; Bubulya, Paula A (2014) The role of SON in splicing, development, and disease. Wiley Interdiscip Rev RNA 5:637-46|
|Lu, Xinyi; Göke, Jonathan; Sachs, Friedrich et al. (2013) SON connects the splicing-regulatory network with pluripotency in human embryonic stem cells. Nat Cell Biol 15:1141-1152|
|Varia, Sapna; Potabathula, Divya; Deng, Zhihui et al. (2013) Btf and TRAP150 have distinct roles in regulating subcellular mRNA distribution. Nucleus 4:229-40|
|Peng, Hong-Juan; Henkels, Karen M; Mahankali, Madhu et al. (2011) The dual effect of Rac2 on phospholipase D2 regulation that explains both the onset and termination of chemotaxis. Mol Cell Biol 31:2227-40|
|Sharma, Alok; Markey, Michael; Torres-Munoz, Keshia et al. (2011) Son maintains accurate splicing for a subset of human pre-mRNAs. J Cell Sci 124:4286-98|
|Tripathi, Vidisha; Ellis, Jonathan D; Shen, Zhen et al. (2010) The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol Cell 39:925-38|
|Sharma, Alok; Takata, Hideaki; Shibahara, Kei-ichi et al. (2010) Son is essential for nuclear speckle organization and cell cycle progression. Mol Biol Cell 21:650-63|
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