Biology subjects

Corey, D. R.

Publications and source records attributed to Corey, D. R..

9 recordsLinked to original sources

bioRxiv2025

g.nome, A Transparent Bioinformatics Pipeline that Enables Differential Expression and Alternative Splicing Analysis by Non-Computational Biologists

Reproducibility and accessibility are cardinal principles in the rapidly evolving field of bioinformatics. As the collection of biological data grows, proper use of pipelines to analyze datasets can become a bottleneck restricting efficient analysis. Biologists who collect data and test hypotheses may not have strong computational backgrounds and may not be able to fully understand the underlying strengths and weaknesses of computational approaches or fully exploit their data. Some data may be misunderstood and, perhaps more importantly, critical findings may remain unobserved. High throughput RNA sequencing (RNAseq) has advanced our understanding of transcriptomics across diverse applications. Here we introduce g.nome, a bioinformatics platform that integrates contemporary tools necessary for independent analysis. A user-friendly graphical interface simplifies running jobs and allows simplified analysis of different datasets by non-bioinformaticians. g.nome was used to analyze the consequences of localizing the critical RNAi factor argonaute (AGO) to nuclei of colorectal cancer cell line HCT116. Analysis using the pipeline facilitated the straightforward identification of splicing changes and the prioritization of these splicing changes for validation and further experimental analysis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/652286v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@f0fa7org.highwire.dtl.DTLVardef@ccac5borg.highwire.dtl.DTLVardef@147c331org.highwire.dtl.DTLVardef@5fe66c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Nuclear Argonaute:miRNA complexes recognize target sequences within chromatin and silence gene expression

RNA interference (RNAi) in mammalian cells involves recognition of mRNA in the cytoplasm and inhibition of translation. Both protein RNAi factors and miRNAs, however, are present in mammalian cell nuclei. It is unclear how this nuclear localization affects endogenous gene expression. Here, we use chimeric eCLIP to identify complexes of Argonaute 2 (AGO2) and miRNAs. We identify the most abundant miRNAs associated with chromatin and their chromatin-associated RNA targets. Chimeric eCLIP revealed that High mobility group AT-Hook A (HMGA2) was the most compelling target for miRNA-mediated gene binding. There are four confirmed let-7 miRNA sites within the 3-UTR in the cytoplasm or nucleus and three within chromatin-associated RNA. The expression of mature HMGA2 mRNA was repressed by let-7 in the cytoplasm and nucleus. let-7 had little effect splicing or transcription. Our data validate chimeric eCLIP as a powerful method for experimentally identifying promising miRNA:RNA interactions. Rather than a solely cytoplasmic event, binding of RNAi factors to mRNA targets may begin in the nucleus through a mechanism that can reduce RNA levels in both the cytoplasm and the nucleus. miRNA-mediated silencing of mRNAs may be influenced by both nuclear and cytoplasmic interactions.

biochemistry↗

bioRxiv2025

Small molecule inhibition of CPSF3 impacts R-loop distribution

R-loops are three-stranded nucleic acid structures consisting of an RNA/DNA hybrid and a displaced strand of DNA. These structures have been implicated in a variety of regulatory cellular processes. Their untimed or excess accumulation, however, can cause genomic instability and induce DNA damage. Most R-loops form co-transcriptionally when the nascent transcript reanneals to unwound DNA duplex. Changes in the rate of transcription have the potential to impact R-loop formation, and compounds that modulate R-loop formation would be useful molecular tools and therapeutic leads. Cleavage and Polyadenylation Specific Factor 3 (CPSF3) recognizes the pre-mRNA 3 cleavage site, cleaves the transcript prior to polyadenylation, and has been linked to R-loop formation. Inhibition of CPSF3 has been found to induce transcriptional readthrough and cell proliferation defects. A previous report suggested that inhibition of CPSF3 with a small molecule causes a global increase in R-loop abundance. Here we test the impact of YT-II-100, a novel inhibitor of CPSF3. We find that addition of YT-II-100 increases global R-loop formation but does not change R-loop formation at specific genes that are normally used as positive controls for R-loop formation. We performed parallel assays using previously reported compound JTE-607 and observed similar results. Our data emphasize the need for cautious interpretation of experiments using JTE-607 and YT-II-100. There may be different mechanisms of R-loop formation depending on gene loci, with the control of R-loop formation at some genes diverging from the regulation of global R-loop formation.

biochemistry↗

bioRxiv2025

Modulation of TTR Gene Expression in the Eye using Modified Duplex RNAs

Small interfering RNAs (siRNAs) are a proven therapeutic approach for controlling gene expression in the liver. Expanding the clinical potential of RNA interference (RNAi) requires developing strategies to enhance delivery to extra-hepatic tissues. In this study we examine inhibiting transthyretin (TRR) gene expression by short interfering RNAs (siRNAs) in the eye. Anti-TTR siRNAs have been developed as successful drugs to treat TTR amyloidosis. When administered systemically, anti-TTR siRNAs alleviate symptoms by blocking TTR expression in the liver. However, TTR amyloidosis also affects the eye, suggesting a need for reducing ocular TTR gene expression. Here, we demonstrate that C5 and 2-O-linked lipid-modified siRNAs formulated in saline can inhibit TTR expression in the eye when administered locally by intravitreal (IVT) injection. Modeling suggests that length and accessibility of the lipid chains contributes to in vivo silencing. GalNAc modified anti-dsRNAs also inhibit TTR expression, albeit less potently. These data support lipid modified siRNAs as an approach to treating the ocular consequences of TTR amyloidosis. Inhibition of TTR expression throughout the eye demonstrates that lipid-siRNA conjugates have the potential to be a versatile platform for ocular drug discovery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/642595v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1e9ad27org.highwire.dtl.DTLVardef@67067borg.highwire.dtl.DTLVardef@1da7eadorg.highwire.dtl.DTLVardef@1b2a552_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Nuclear Localization of Argonaute is affected by Cell Density and May Relieve Repression by microRNAs

Argonaute protein is associated with post-transcriptional control of cytoplasmic gene expression through miRNA-induced silencing complexes (miRISC). Specific cellular and environmental conditions can trigger AGO protein to accumulate in the nucleus. Localization of AGO is central to understanding miRNA action, yet the consequences of AGO being in the nucleus are undefined. We show nuclear enrichment of AGO2 in HCT116 cells grown in two-dimensional culture to high density, HCT116 cells grown in three-dimensional tumor spheroid culture, and human colon tumors. The shift in localization of AGO2 from cytoplasm to nucleus de-represses cytoplasmic AGO2-eCLIP targets that were candidates for canonical regulation by miRISC. Constitutive nuclear localization of AGO2 using an engineered nuclear localization signal increases cell migration. Critical RNAi factors also affect the localization of AGO2. Knocking out an enzyme essential for miRNA biogenesis, DROSHA, depletes mature miRNAs and restricts AGO2 localization to the cytoplasm, while knocking out the miRISC scaffolding protein, TNRC6, results in nuclear localization of AGO2. These data suggest that AGO2 localization and miRNA activity can be regulated depending on environmental conditions, expression of mature miRNAs, and expression of miRISC cofactors. Localization and expression of core miRISC protein machinery should be considered when investigating the roles of miRNAs.

biochemistry↗

bioRxiv2022

Prioritizing Annotated miRNAs: Only a Small Percentage are Candidates for Biological Regulation

The potential for miRNAs to regulate gene expression remains controversial. DROSHA initiates the biogenesis of miRNAs while Argonaute (AGO) and TNRC6 proteins form complexes with miRNAs that recognize RNA. Here we investigate the fate of miRNAs in the absence of critical RNAi protein factors. Knockout of DROSHA expression reduced levels of some miRNAs, but not others. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of miRNAs. Quantitative analysis indicates compensation to maintain the overall pool of AGO after knockout of AGO variants. Evaluation of miRNA binding to AGO proteins revealed that association between AGO and miRNAs was similar for AGO1 - 4. Contrary to the assumptions underlying many peer-reviewed reports, not all annotated miRNAs have equal potential as biological regulators. Cellular abundance, DROSHA dependence, and physical association with AGO must be considered when forming hypotheses related to their function. Our data prioritize sixty miRNAs - under two percent of the overall annotated miRNA repertoire - as being most likely to function as robust gene regulators. Our approach will facilitate identifying biologically active miRNAs.

biochemistry↗

bioRxiv2021

Impact of scaffolding protein TNRC6 paralogs on gene expression and splicing

TNRC6 is a scaffolding protein that bridges interactions between small RNAs, argonaute (AGO) protein, and effector proteins to control gene expression. There are three paralogs in mammalian cells, TNRC6A, TNRC6B, and TNRC6C. These paralogs have ~40% amino acid sequence identity and the extent of their unique or redundant functions is unclear. Here, we use knockout cell lines, enhanced crosslinking immunoprecipitation (eCLIP), and high-throughput RNA sequencing (RNAseq) to explore the roles of TNRC6 paralogs in RNA-mediated control of gene expression. We find that that the paralogs are largely functionally redundant and changes in levels of gene expression are well-correlated with those observed in AGO knockout cell lines. Splicing changes observed in AGO knockout cell lines are observed in TNRC6 knockout cells. These data further define the roles of the TNRC6 isoforms as part of the RNA interference (RNAi) machinery.

molecular biology↗

bioRxiv2021

Argonaute Binding within Human Nuclear RNA and its Impact on Alternative Splicing

Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation (eCLIP) in combination with RNA sequencing (RNAseq) and multiple AGO knockout cell lines, we mapped AGO2 protein binding sites within nuclear RNA. The strongest AGO2 binding sites were mapped to micro RNAs (miRNAs). The most abundant miRNAs were distributed similarly between cytoplasm and nucleus, providing no evidence for mechanisms that facilitate localization of miRNAs in one compartment versus the other. Beyond miRNAs, most statistically-significant AGO2 binding was within introns. Splicing changes were confirmed by RT-PCR and were recapitulated by synthetic duplex RNAs and miRNA mimics complementary to the sites of AGO2 binding. These data support the hypothesis that miRNAs can control gene splicing. While nuclear RNAi proteins have the potential to be a natural regulatory mechanism, careful study will be necessary to identify critical RNA drivers of normal physiology and disease.

molecular biology↗

bioRxiv2020

Argonaute Binding within 3'-Untranslated Regions Does Not Predict Gene Repression

Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: 1) Knockout of argonaute (AGO) variants; 2) RNA sequencing analysis of gene expression changes; and 3) Crosslinking Immunoprecipitation Sequencing (CLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2, and AGO3 are necessary to achieve full impact on gene expression. CLIP-seq reveals several hundred significant AGO2 associations within the 3-untranslated regions of cytoplasmic transcripts. The standard mechanism of miRNA action would suggest that these associations repress gene expression. Contrary to this expectation, clusters are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased gene expression in wild-type versus knock out cells, including the strongest cluster within the MYC 3-UTR. Our results suggest that assumptions about miRNA action should be re-examined.

molecular biology↗