Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated. Taken together, these observations suggest a novel role for some unannotated RNAs as primary transcripts for the production of short RNAs. Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes. These data support a highly interleaved organization of the human transcriptome.

The molecular mechanisms underlying pluripotency and lineage specification from embryonic stem cells (ESCs) are largely unclear. Differentiation pathways may be determined by the targeted activation of lineage-specific genes or by selective silencing of genome regions. Here we show that the ESC genome is transcriptionally globally hyperactive and undergoes large-scale silencing as cells differentiate. Normally silent repeat regions are active in ESCs, and tissue-specific genes are sporadically expressed at low levels. Whole-genome tiling arrays demonstrate widespread transcription in coding and noncoding regions in ESCs, whereas the transcriptional landscape becomes more discrete as differentiation proceeds. The transcriptional hyperactivity in ESCs is accompanied by disproportionate expression of chromatin-remodeling genes and the general transcription machinery. We propose that global transcription is a hallmark of pluripotent ESCs, contributing to their plasticity, and that lineage specification is driven by reduction of the transcribed portion of the genome.

Regulation of mRNA turnover is an important cellular strategy for posttranscriptional control of gene expression, mediated by the interplay of cis-acting sequences and associated trans-acting factors. Pub1p, an ELAV-like yeast RNA-binding protein with homology to T-cell internal antigen 1 (TIA-1)/TIA-1-related protein (TIAR), is an important modulator of the decay of two known classes of mRNA. Our goal in this study was to determine the range of mRNAs whose stability is dependent on Pub1p, as well as to identify specific transcripts that directly bind to this protein. We have examined global mRNA turnover in isogenic PUB1 and pub1⌬ strains through gene expression analysis and demonstrate that 573 genes exhibit a significant reduction in half-life in a pub1⌬ strain. We also examine the binding specificity of Pub1p using affinity purification followed by microarray analysis to comprehensively distinguish between direct and indirect targets and find that Pub1p significantly binds to 368 cellular transcripts. Among the Pub1p-associated mRNAs, 53 transcripts encoding proteins involved in ribosomal biogenesis and cellular metabolism are selectively destabilized in the pub1⌬ strain. In contrast, genes involved in transporter activity demonstrate association with Pub1p but display no measurable changes in transcript stability. Characterization of two candidate genes, SEC53 and RPS16B, demonstrate that both Pub1p-dependent regulation of stability and Pub1p binding require 3 untranslated regions, which harbor distinct sequence motifs. These results suggest that Pub1p binds to discrete subsets of cellular transcripts and posttranscriptionally regulates their expression at multiple levels.Gene expression in eukaryotes is a highly diverse process, involving regulation at both transcriptional and posttranscriptional levels (47, 70). The process of mRNA turnover is an important posttranscriptional control point that helps to modulate the cellular abundance of a transcript. A large number of clinically relevant transcripts exhibit regulated decay in response to cellular signals, and the deregulation of their decay rates directly correlates with disease states (58,66,70). In the yeast Saccharomyces cerevisiae, the principal mRNA degradation pathway initiates with the removal of the poly(A) tail, followed by either decapping and 5Ј33Ј exonucleolytic decay or 3Ј35Ј exosome-mediated degradation (10, 64). In addition to this pathway, aberrant mRNAs harboring premature termination codons are degraded by an alternate nonsense-mediated decay (NMD) pathway that functions to ensure quality control of gene expression. NMD is initiated when a premature termination codon is recognized during translation termination and stimulates rapid deadenylation-independent decapping, followed by 5Ј33Јdegradation of the mRNA (4). Messages undergoing leaky scanning (69) or harboring upstream open reading frames (50, 59) represent some naturally occurring substrates that decay through this pathway.The turnover of mRNAs is mediated by the interplay between...

Many eukaryotic mRNAs exhibit regulated decay in response to cellular signals. AU-rich elements (AREs) identified in the 3 untranslated region (3-UTR) of several such mRNAs play a critical role in controlling the half-lives of these transcripts. The yeast ARE-containing mRNA, MFA2, has been studied extensively and is degraded by a deadenylation-dependent mechanism. However, the trans-acting factors that promote the rapid decay of MFA2 have not been identified. Our results suggest that the chaperone protein Hsp70, encoded by the SSA family of genes, is involved in modulating MFA2 mRNA decay. MFA2 is specifically stabilized in a strain bearing a temperature-sensitive mutation in the SSA1 gene. Furthermore, an AU-rich region within the 3-UTR of the message is both necessary and sufficient to confer this regulation. Stabilization occurs as a result of slower deadenylation in the ssa1 ts strain, suggesting that Hsp70 is required for activation of the turnover pathway.Gene expression is a highly controlled process involving regulation at both transcriptional and posttranscriptional levels. In recent years mRNA turnover has emerged as an important target for the regulation of gene expression (33,54). A large number of mRNAs encoding cytokines, growth factors, and proto-oncogenes display regulated decay in response to external signals (44, 50). Hence, understanding the pathways regulating transcript stability is of critical importance. Selective mRNA degradation is mediated by a number of different cisacting sequences, including the most-investigated class, called AU-rich elements (AREs), present in the 3Ј untranslated region (3Ј-UTR) of a variety of mammalian and yeast mRNAs (44,49,54). Recent experiments have shown that the pathways and factors involved in ARE-mediated mRNA decay are conserved between yeast and higher eukaryotes, making yeast an ideal system for the study of this phenomenon (49).The ARE is a stability determinant whose sequence is loosely defined and ranges in size from 50 to 150 nucleotides. These elements are typically found in the 3Ј-UTR and contain one or more copies of the pentameric sequence AUUUA flanked by a high content of U's and A's (8, 47). An important feature of many AREs is that they modulate the stability of transcripts in response to cellular stimuli. They can cause instability under some conditions by enhancing the rate of removal of the poly(A) tail and the subsequent degradation of the body of the transcript (7, 38, 48). In contrast, under stabilizing conditions AREs can inhibit the decay process (see references 49 and 54 and references therein). In yeast, at least two classes of ARE-containing mRNAs have been identified, represented by the MFA2 and TIF51A/HYP2 transcripts (49). In both cases decay proceeds through poly(A) tail shortening followed by decapping and 5Ј33Ј exonucleolytic decay (36, 49). The stability of the TIF51A transcript is modulated in response to changes in carbon source (11,49). The mRNA is stable in cells grown in glucose and unstable in cells grown under nong...

The transcriptional landscape in embryonic stem cells (ESCs) and during ESC differentiation has received considerable attention, albeit mostly confined to the polyadenylated fraction of RNA, whereas the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in ESCs using a combination of whole-genome tiling arrays and deep sequencing technologies. In addition to identifying previously characterized and new non-coding RNA members, we describe a group of novel conserved RNAs (snacRNAs: small NPA conserved), some of which are differentially expressed between ESC and neuronal progenitor cells, providing the first evidence of a novel group of potentially functional NPA RNA involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal small nuclear RNAs, which are NPA, are almost completely absent in ESCs and are upregulated in differentiation. Finally, we show differential processing of the minor intron of the polycomb group gene Eed. Our data suggest that NPA RNA, both known and novel, play important roles in ESCs.

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