Beta Cell 5′-Shifted isomiRs Are Candidate Regulatory Hubs in Type 2 Diabetes

Jeanette Baran-Gale,Emily E Fannin,Praveen Sethupathy,C Lisa Kurtz

doi:10.1371/journal.pone.0073240

Jeanette Baran-Gale, Emily E Fannin + Show 2 more

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https://doi.org/10.1371/journal.pone.0073240

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Journal: PLoS ONE	Publication Date: Sep 9, 2013
Citations: 114	License type: CC BY 4.0

Affiliation: University of North Carolina at Chapel Hill

Abstract

Next-generation deep sequencing of small RNAs has unveiled the complexity of the microRNA (miRNA) transcriptome, which is in large part due to the diversity of miRNA sequence variants (“isomiRs”). Changes to a miRNA’s seed sequence (nucleotides 2–8), including shifted start positions, can redirect targeting to a dramatically different set of RNAs and alter biological function. We performed deep sequencing of small RNA from mouse insulinoma (MIN6) cells (widely used as a surrogate for the study of pancreatic beta cells) and developed a bioinformatic analysis pipeline to profile isomiR diversity. Additionally, we applied the pipeline to recently published small RNA-seq data from primary human beta cells and whole islets and compared the miRNA profiles with that of MIN6. We found that: (1) the miRNA expression profile in MIN6 cells is highly correlated with those of primary human beta cells and whole islets; (2) miRNA loci can generate multiple highly expressed isomiRs with different 5′-start positions (5′-isomiRs); (3) isomiRs with shifted start positions (5′-shifted isomiRs) are highly expressed, and can be as abundant as their unshifted counterparts (5′-reference miRNAs). Finally, we identified 10 beta cell miRNA families as candidate regulatory hubs in a type 2 diabetes (T2D) gene network. The most significant candidate hub was miR-29, which we demonstrated regulates the mRNA levels of several genes critical to beta cell function and implicated in T2D. Three of the candidate miRNA hubs were novel 5′-shifted isomiRs: miR-375+1, miR-375-1 and miR-183-5p+1. We showed by in silico target prediction and in vitro transfection studies that both miR-375+1 and miR-375-1 are likely to target an overlapping, but distinct suite of beta cell genes compared to canonical miR-375. In summary, this study characterizes the isomiR profile in beta cells for the first time, and also highlights the potential functional relevance of 5′-shifted isomiRs to T2D.

Highlights

MiRNAs are short regulatory RNAs that are processed from variable length primary transcripts through consecutive ribonuclease-mediated cleavage events [1,2]. miRNAs guide and tether the RNA induced silencing complex (RISC) to specific RNAs in order to regulate their stability and/or translation [3]
In this study we developed an in-house bioinformatic analysis pipeline to characterize isomiR diversity, and applied this method to study isomiR expression in the MIN6 cell line, primary human beta cells and islets
We found that (1) the miRNA expression profile in the MIN6 cell line is highly correlated with that of the primary human beta cells, (2) miRNA loci can be classified as either homogeneous or heterogeneous, (3) 59-shifted isomiRs can be as abundant as their 59reference counterparts, and (4) there are seven 59-shifted isomiRs highly expressed in MIN6 cells that are abundant in human beta cells and islets

Summary

Introduction

MiRNAs are short regulatory RNAs that are processed from variable length primary transcripts through consecutive ribonuclease-mediated cleavage events [1,2]. miRNAs guide and tether the RNA induced silencing complex (RISC) to specific RNAs in order to regulate their stability and/or translation [3]. Similar to protein coding genes, miRNAs are present in multiple isoforms, called isomiRs [8,9,10]. IsomiRs are sequence variants, generated from a single miRNA locus, that consist of one or both of two types of variations: templated and non-templated [9,11,12] (Fig. 1). Templated variants match the genomic sequence, but have differing 59-start and/or 39-end positions, likely due to processing heterogeneity by Drosha/Dicer [1,2] and/ or exonuclease-mediated nucleotide trimming [13,14]. Nontemplated isomiRs are diverged from the genomic sequence due to post-transcriptional enzymatic processes that add, remove, or edit specific nucleotides [10]. The most prevalent form of RNA editing is the adenosine-toinosine edit, which is mediated by the double-stranded RNA adenosine deaminase (ADAR) family of enzymes [16]

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