Publications by authors named "Rakesh Chatrikhi"

5 Publications

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Reciprocal regulation of hnRNP C and CELF2 through translation and transcription tunes splicing activity in T cells.

Nucleic Acids Res 2020 06;48(10):5710-5719

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

RNA binding proteins (RBPs) frequently regulate the expression of other RBPs in mammalian cells. Such cross-regulation has been proposed to be important to control networks of coordinated gene expression; however, much remains to be understood about how such networks of cross-regulation are established and what the functional consequence is of coordinated or reciprocal expression of RBPs. Here we demonstrate that the RBPs CELF2 and hnRNP C regulate the expression of each other, such that depletion of one results in reduced expression of the other. Specifically, we show that loss of hnRNP C reduces the transcription of CELF2 mRNA, while loss of CELF2 results in decreased efficiency of hnRNP C translation. We further demonstrate that this reciprocal regulation serves to fine tune the splicing patterns of many downstream target genes. Together, this work reveals new activities of hnRNP C and CELF2, provides insight into a previously unrecognized gene regulatory network, and demonstrates how cross-regulation of RBPs functions to shape the cellular transcriptome.
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http://dx.doi.org/10.1093/nar/gkaa295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261192PMC
June 2020

RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery.

Cell Rep 2019 09;28(11):2795-2806.e3

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The 3' UTR (UTR) of human mRNAs plays a critical role in controlling protein expression and function. Importantly, 3' UTRs of human messages are not invariant for each gene but rather are shaped by alternative polyadenylation (APA) in a cell state-dependent manner, including in response to T cell activation. However, the proteins and mechanisms driving APA regulation remain poorly understood. Here we show that the RNA-binding protein CELF2 controls APA of its own message in a signal-dependent manner by competing with core enhancers of the polyadenylation machinery for binding to RNA. We further show that CELF2 binding overlaps with APA enhancers transcriptome-wide, and almost half of 3' UTRs that undergo T cell signaling-induced APA are regulated in a CELF2-dependent manner. These studies thus reveal CELF2 to be a critical regulator of 3' UTR identity in T cells and demonstrate an additional mechanism for CELF2 in regulating polyadenylation site choice.
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http://dx.doi.org/10.1016/j.celrep.2019.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752737PMC
September 2019

SF1 Phosphorylation Enhances Specific Binding to U2AF and Reduces Binding to 3'-Splice-Site RNA.

Biophys J 2016 Dec;111(12):2570-2586

Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York. Electronic address:

Splicing factor 1 (SF1) recognizes 3' splice sites of the major class of introns as a ternary complex with U2AF and U2AF splicing factors. A conserved SPSP motif in a coiled-coil domain of SF1 is highly phosphorylated in proliferating human cells and is required for cell proliferation. The UHM kinase 1 (UHMK1), also called KIS, double-phosphorylates both serines of this SF1 motif. Here, we use isothermal titration calorimetry to demonstrate that UHMK1 phosphorylation of the SF1 SPSP motif slightly enhances specific binding of phospho-SF1 to its cognate U2AF protein partner. Conversely, quantitative fluorescence anisotropy RNA binding assays and isothermal titration calorimetry experiments establish that double-SPSP phosphorylation reduces phospho-SF1 and phospho-SF1-U2AF binding affinities for either optimal or suboptimal splice-site RNAs. Domain-substitution and mutagenesis experiments further demonstrate that arginines surrounding the phosphorylated SF1 loop are required for cooperative 3' splice site recognition by the SF1-U2AF complex (where cooperativity is defined as a nonadditive increase in RNA binding by the protein complex relative to the individual proteins). In the context of local, intracellular concentrations, the subtle effects of SF1 phosphorylation on its associations with U2AF and splice-site RNAs are likely to influence pre-mRNA splicing. However, considering roles for SF1 in pre-mRNA retention and transcriptional repression, as well as in splicing, future comprehensive investigations are needed to fully explain the requirement for SF1 SPSP phosphorylation in proliferating human cells.
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http://dx.doi.org/10.1016/j.bpj.2016.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192697PMC
December 2016

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival.

PLoS Genet 2016 Oct 24;12(10):e1006384. Epub 2016 Oct 24.

Cancer Biology Section, Cancer Genetics Branch, National Human Genome Research Institute, Bethesda, United States Of America.

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3' splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3' splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele.
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http://dx.doi.org/10.1371/journal.pgen.1006384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5077151PMC
October 2016

An extended U2AF(65)-RNA-binding domain recognizes the 3' splice site signal.

Nat Commun 2016 Mar 8;7:10950. Epub 2016 Mar 8.

Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.

How the essential pre-mRNA splicing factor U2AF(65) recognizes the polypyrimidine (Py) signals of the major class of 3' splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF(65)-RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF(65) inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF(65) linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3' terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF(65) RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.
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http://dx.doi.org/10.1038/ncomms10950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786784PMC
March 2016