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      Experimental Verification and Evolutionary Origin of 5′-UTR Polyadenylation Sites in Arabidopsis thaliana

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          Abstract

          Messenger RNA (mRNA) polyadenylation is an indispensable step during post-transcriptional pre-mRNA processing for most genes in eukaryotes. The usage of one poly(A) site over another is known as alternative polyadenylation (APA). APA has been implicated in gene expression regulation through its role of selecting the ends of a transcript. Recent studies of polyadenylation profiles in the Arabidopsis database unexpectedly predicted that a portion of the poly(A) sites are located in the 5′-UTR, which remains to be experimentally verified. We selected 16 genes from a dataset of 744, based on criteria designed to minimize problems in interpretation. Here, we experimentally verify 5′-UTR-APA in Arabidopsis for 10 of the 16 selected genes, and show for the first time existence of independent polyadenylated 5′-UTR transcripts, arising due to alternative polyadenylation. We used 3′-RACE and sequencing to validate poly(A) sites and northern blot to show that the observed short upstream transcripts do not arise from the 3′-end of a previously unrecognized convergent gene. Evidence is reported showing that two of the independent upstream open reading frame (uORF) transcripts studied, one containing a complex dual uORF, very likely arose by exon shuffling following duplication of the 5′-end from the downstream major open reading frame (mORF). Finally, results are presented to show that the uORF in this gene may encode two short functional proteins, based on observation of amino acid sequence conservation encoded by the dual uORFs.

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          The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance.

          Mitochondria are known to be functional organelles, but their role as a signaling unit is increasingly being appreciated. The identification of a short open reading frame (sORF) in the mitochondrial DNA (mtDNA) that encodes a signaling peptide, humanin, suggests the possible existence of additional sORFs in the mtDNA. Here we report a sORF within the mitochondrial 12S rRNA encoding a 16-amino-acid peptide named MOTS-c (mitochondrial open reading frame of the 12S rRNA-c) that regulates insulin sensitivity and metabolic homeostasis. Its primary target organ appears to be the skeletal muscle, and its cellular actions inhibit the folate cycle and its tethered de novo purine biosynthesis, leading to AMPK activation. MOTS-c treatment in mice prevented age-dependent and high-fat-diet-induced insulin resistance, as well as diet-induced obesity. These results suggest that mitochondria may actively regulate metabolic homeostasis at the cellular and organismal level via peptides encoded within their genome.
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            Molecular mechanisms of eukaryotic pre-mRNA 3′ end processing regulation

            Messenger RNA (mRNA) 3′ end formation is a nuclear process through which all eukaryotic primary transcripts are endonucleolytically cleaved and most of them acquire a poly(A) tail. This process, which consists in the recognition of defined poly(A) signals of the pre-mRNAs by a large cleavage/polyadenylation machinery, plays a critical role in gene expression. Indeed, the poly(A) tail of a mature mRNA is essential for its functions, including stability, translocation to the cytoplasm and translation. In addition, this process serves as a bridge in the network connecting the different transcription, capping, splicing and export machineries. It also participates in the quantitative and qualitative regulation of gene expression in a variety of biological processes through the selection of single or alternative poly(A) signals in transcription units. A large number of protein factors associates with this machinery to regulate the efficiency and specificity of this process and to mediate its interaction with other nuclear events. Here, we review the eukaryotic 3′ end processing machineries as well as the comprehensive set of regulatory factors and discuss the different molecular mechanisms of 3′ end processing regulation by proposing several overlapping models of regulation.
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              Genome-wide landscape of polyadenylation in Arabidopsis provides evidence for extensive alternative polyadenylation.

              Alternative polyadenylation (APA) has been shown to play an important role in gene expression regulation in animals and plants. However, the extent of sense and antisense APA at the genome level is not known. We developed a deep-sequencing protocol that queries the junctions of 3'UTR and poly(A) tails and confidently maps the poly(A) tags to the annotated genome. The results of this mapping show that 70% of Arabidopsis genes use more than one poly(A) site, excluding microheterogeneity. Analysis of the poly(A) tags reveal extensive APA in introns and coding sequences, results of which can significantly alter transcript sequences and their encoding proteins. Although the interplay of intron splicing and polyadenylation potentially defines poly(A) site uses in introns, the polyadenylation signals leading to the use of CDS protein-coding region poly(A) sites are distinct from the rest of the genome. Interestingly, a large number of poly(A) sites correspond to putative antisense transcripts that overlap with the promoter of the associated sense transcript, a mode previously demonstrated to regulate sense gene expression. Our results suggest that APA plays a far greater role in gene expression in plants than previously expected.
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                Author and article information

                Contributors
                Journal
                Front Plant Sci
                Front Plant Sci
                Front. Plant Sci.
                Frontiers in Plant Science
                Frontiers Media S.A.
                1664-462X
                05 July 2018
                2018
                : 9
                : 969
                Affiliations
                Program in Cell Molecular and Structural Biology, Department of Biology, Miami University , Oxford, OH, United States
                Author notes

                Edited by: Fulvio Cruciani, Sapienza Università di Roma, Italy

                Reviewed by: Xusheng Wang, St. Jude Children's Research Hospital, United States; René Massimiliano Marsano, Università degli Studi di Bari Aldo Moro, Italy

                *Correspondence: Jack C. Vaughn vaughnjc@ 123456miamioh.edu

                This article was submitted to Evolutionary and Population Genetics, a section of the journal Frontiers in Plant Science

                Article
                10.3389/fpls.2018.00969
                6041940
                57928356-37eb-4128-95f2-90dde1d7e59d
                Copyright © 2018 Zhu and Vaughn.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 31 December 2017
                : 15 June 2018
                Page count
                Figures: 8, Tables: 2, Equations: 0, References: 43, Pages: 12, Words: 7603
                Funding
                Funded by: National Institutes of Health 10.13039/100000002
                Award ID: 1-R15-GM093895-01
                Categories
                Plant Science
                Original Research

                Plant science & Botany
                5′-utr,alternative polyadenylation,exon evolution,exon shuffling,uorf
                Plant science & Botany
                5′-utr, alternative polyadenylation, exon evolution, exon shuffling, uorf

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