Insights into the roles of local translation from the axonal transcriptome

Much of our knowledge on the roles of intra-axonal translation derives from the characterization of a small number of individual mRNAs that were found to be localized in axons. However, two recent studies, using large-scale approaches to provide a more comprehensive characterization of the axonal transcriptome, have led to the discovery of thousands of axonal mRNAs. The apparent abundance of mRNAs in axons raises the possibility that local translation has many more functions than previously thought. Here, we review the recent studies that have profiled axonal mRNAs and discuss how the identification of axonal transcripts might point to unappreciated roles for local translation in axons

Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons

SummaryMethylation of the N6 position of adenosine (m6A) is a posttranscriptional modification of RNA with poorly understood prevalence and physiological relevance. The recent discovery that FTO, an obesity risk gene, encodes an m6A demethylase implicates m6A as an important regulator of physiological processes. Here, we present a method for transcriptome-wide m6A localization, which combines m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq). We use this method to identify mRNAs of 7,676 mammalian genes that contain m6A, indicating that m6A is a common base modification of mRNA. The m6A modification exhibits tissue-specific regulation and is markedly increased throughout brain development. We find that m6A sites are enriched near stop codons and in 3′ UTRs, and we uncover an association between m6A residues and microRNA-binding sites within 3′ UTRs. These findings provide a resource for identifying transcripts that are substrates for adenosine methylation and reveal insights into the epigenetic regulation of the mammalian transcriptome

Global profiling of stimulus-induced polyadenylation in cells using a poly(A) trap

Polyadenylation of mRNA leads to increased protein expression in response to diverse stimuli, but it is difficult to identify mRNAs that become polyadenylated in living cells. Here we describe a click chemistry-compatible nucleoside analog that is selectively incorporated into poly(A) tails of transcripts in cells. Next-generation sequencing of labeled mRNAs enables a transcriptome-wide profile of polyadenylation and provides insights into the mRNA sequence elements that are correlated with polyadenylation

m^6A RNA methylation promotes XIST-mediated transcriptional repression

The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N^6-methyladenosine (m^6A) residues—a reversible base modification of unknown function in long non-coding RNAs. We show that m^6A formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m^6A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m^6A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m^6A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m^6A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m^6A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m^6A. These data reveal a pathway of m^6A formation and recognition required for XIST-mediated transcriptional repression

Metazoan tRNA introns generate stable circular RNAs in vivo

We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating “designer” circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science

m^6A RNA methylation promotes XIST-mediated transcriptional repression

The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N^6-methyladenosine (m^6A) residues—a reversible base modification of unknown function in long non-coding RNAs. We show that m^6A formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m^6A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m^6A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m^6A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m^6A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m^6A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m^6A. These data reveal a pathway of m^6A formation and recognition required for XIST-mediated transcriptional repression