Publications by authors named "Sean P McClory"

6 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

HnRNP L represses cryptic exons.

RNA 2018 06 26;24(6):761-768. Epub 2018 Mar 26.

Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA.

The fidelity of RNA splicing is regulated by a network of splicing enhancers and repressors, although the rules that govern this process are not yet fully understood. One mechanism that contributes to splicing fidelity is the repression of nonconserved cryptic exons by splicing factors that recognize dinucleotide repeats. We previously identified that TDP-43 and PTBP1/PTBP2 are capable of repressing cryptic exons utilizing UG and CU repeats, respectively. Here we demonstrate that hnRNP L () also represses cryptic exons by utilizing exonic CA repeats, particularly near the 5'SS. We hypothesize that hnRNP L regulates CA repeat repression for both cryptic exon repression and developmental processes such as T cell differentiation.
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http://dx.doi.org/10.1261/rna.065508.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959245PMC
June 2018

The human PMR1 endonuclease stimulates cell motility by down regulating miR-200 family microRNAs.

Nucleic Acids Res 2016 07 1;44(12):5811-9. Epub 2016 Jun 1.

Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH 43210, USA

The motility of MCF-7 cells increases following expression of a human PMR1 transgene and the current study sought to identify the molecular basis for this phenotypic change. Ensemble and single cell analyses show increased motility is dependent on the endonuclease activity of hPMR1, and cells expressing active but not inactive hPMR1 invade extracellular matrix. Nanostring profiling identified 14 microRNAs that are downregulated by hPMR1, including all five members of the miR-200 family and others that also regulate invasive growth. miR-200 levels increase following hPMR1 knockdown, and changes in miR-200 family microRNAs were matched by corresponding changes in miR-200 targets and reporter expression. PMR1 preferentially cleaves between UG dinucleotides within a consensus YUGR element when present in the unpaired loop of a stem-loop structure. This motif is present in the apical loop of precursors to most of the downregulated microRNAs, and hPMR1 targeting of pre-miRs was confirmed by their loss following induced expression and increase following hPMR1 knockdown. Introduction of miR-200c into hPMR1-expressing cells reduced motility and miR-200 target gene expression, confirming hPMR1 acts upstream of Dicer processing. These findings identify a new role for hPMR1 in the post-transcriptional regulation of microRNAs in breast cancer cells.
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http://dx.doi.org/10.1093/nar/gkw497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937341PMC
July 2016

In-cell SHAPE reveals that free 30S ribosome subunits are in the inactive state.

Proc Natl Acad Sci U S A 2015 Feb 9;112(8):2425-30. Epub 2015 Feb 9.

Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290; and

It was shown decades ago that purified 30S ribosome subunits readily interconvert between "active" and "inactive" conformations in a switch that involves changes in the functionally important neck and decoding regions. However, the physiological significance of this conformational change had remained unknown. In exponentially growing Escherichia coli cells, RNA SHAPE probing revealed that 16S rRNA largely adopts the inactive conformation in stably assembled, mature 30S subunits and the active conformation in translating (70S) ribosomes. Inactive 30S subunits bind mRNA as efficiently as active subunits but initiate translation more slowly. Mutations that inhibited interconversion between states compromised translation in vivo. Binding by the small antibiotic paromomycin induced the inactive-to-active conversion, consistent with a low-energy barrier between the two states. Despite the small energetic barrier between states, but consistent with slow translation initiation and a functional role in vivo, interconversion involved large-scale changes in structure in the neck region that likely propagate across the 30S body via helix 44. These findings suggest the inactive state is a biologically relevant alternate conformation that regulates ribosome function as a conformational switch.
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http://dx.doi.org/10.1073/pnas.1411514112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345610PMC
February 2015

Distinct functional classes of ram mutations in 16S rRNA.

RNA 2014 Apr 26;20(4):496-504. Epub 2014 Feb 26.

During decoding, the ribosome selects the correct (cognate) aminoacyl-tRNA (aa-tRNA) from a large pool of incorrect aa-tRNAs through a two-stage mechanism. In the initial selection stage, aa-tRNA is delivered to the ribosome as part of a ternary complex with elongation factor EF-Tu and GTP. Interactions between codon and anticodon lead to activation of the GTPase domain of EF-Tu and GTP hydrolysis. Then, in the proofreading stage, aa-tRNA is released from EF-Tu and either moves fully into the A/A site (a step termed "accommodation") or dissociates from the ribosome. Cognate codon-anticodon pairing not only stabilizes aa-tRNA at both stages of decoding but also stimulates GTP hydrolysis and accommodation, allowing the process to be both accurate and fast. In previous work, we isolated a number of ribosomal ambiguity (ram) mutations in 16S rRNA, implicating particular regions of the ribosome in the mechanism of decoding. Here, we analyze a representative subset of these mutations with respect to initial selection, proofreading, RF2-dependent termination, and overall miscoding in various contexts. We find that mutations that disrupt inter-subunit bridge B8 increase miscoding in a general way, causing defects in both initial selection and proofreading. Mutations in or near the A site behave differently, increasing miscoding in a codon-anticodon-dependent manner. These latter mutations may create spurious favorable interactions in the A site for certain near-cognate aa-tRNAs, providing an explanation for their context-dependent phenotypes in the cell.
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http://dx.doi.org/10.1261/rna.043331.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964911PMC
April 2014

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection.

RNA 2010 Oct 10;16(10):1925-34. Epub 2010 Aug 10.

Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.

The molecular basis of the induced-fit mechanism that determines the fidelity of protein synthesis remains unclear. Here, we isolated mutations in 16S rRNA that increase the rate of miscoding and stop codon read-through. Many of the mutations clustered along interfaces between the 30S shoulder domain and other parts of the ribosome, strongly implicating shoulder movement in the induced-fit mechanism of decoding. The largest subset of mutations mapped to helices h8 and h14. These helices interact with each other and with the 50S subunit to form bridge B8. Previous cryo-EM studies revealed a contact between h14 and the switch 1 motif of EF-Tu, raising the possibility that h14 plays a direct role in GTPase activation. To investigate this possibility, we constructed both deletions and insertions in h14. While ribosomes harboring a 2-base-pair (bp) insertion in h14 were completely inactive in vivo, those containing a 2-bp deletion retained activity but were error prone. In vitro, the truncation of h14 accelerated GTP hydrolysis for EF-Tu bearing near-cognate aminoacyl-tRNA, an effect that can largely account for the observed miscoding in vivo. These data show that h14 does not help activate EF-Tu but instead negatively controls GTP hydrolysis by the factor. We propose that bridge B8 normally acts to counter inward rotation of the shoulder domain; hence, mutations in h8 and h14 that compromise this bridge decrease the stringency of aminoacyl-tRNA selection.
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http://dx.doi.org/10.1261/rna.2228510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2941101PMC
October 2010