Therese A. Yario

Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5′ UTR as in the 3′ UTR

In animals, microRNAs (miRNAs) bind to the 3′ UTRs of their target mRNAs and interfere with translation, although the exact mechanism of inhibition of protein synthesis remains unclear. Functional miRNA-binding sites in the coding regions or 5′ UTRs of endogenous mRNAs have not been identified. We studied the effect of introducing miRNA target sites into the 5′ UTR of luciferase reporter mRNAs containing internal ribosome entry sites (IRESs), so that potential steric hindrance by a microribonucleoprotein complex would not interfere with the initiation of translation. In human HeLa cells, which express endogenous let-7a miRNA, the translational efficiency of these IRES-containing reporters with 5′ let-7 complementary sites from the Caenorhabditis elegans lin-41 3′ UTR was repressed. Similarly, the IRES-containing reporters were translationally repressed when human Ago2 was tethered to either the 5′ or 3′ UTR. Interestingly, the method of DNA transfection affected our ability to observe miRNA-mediated repression. Our results suggest that association with any position on a target mRNA is mechanistically sufficient for a microribonucleoprotein to exert repression of translation at some step downstream of initiation.

Down-Regulation of a Host MicroRNA by a Herpesvirus saimiri Noncoding RNA

Herpes Virus MiRNA Modulation Viruses use a number of strategies to manipulate the cells of their host to ensure a successful infection. Herpesvirus saimiri (HVS) generates highly conserved small noncoding RNAs HSUR 1 and HSUR 2, which modulate expression of a number of proteins in infected primate T cells. Cazalla et al. (p. 1563; see the Perspective by Pasquinelli) observed complementarity between HSUR sequences and the seed regions of three different miRNAs—miR-142-3p, miR-27, and miR-16—and found that these HSURs could bind to the miRNAs. Furthermore, the level of mature miR-27 was modulated by binding to HSUR 1, which targeted the miRNA for degradation. A viral noncoding RNA regulates the level of a complementary host-cell microRNA. T cells transformed by Herpesvirus saimiri express seven viral U-rich noncoding RNAs of unknown function called HSURs. We noted that conserved sequences in HSURs 1 and 2 constitute potential binding sites for three host-cell microRNAs (miRNAs). Coimmunoprecipitation experiments confirmed that HSURs 1 and 2 interact with the predicted miRNAs in virally transformed T cells. The abundance of one of these miRNAs, miR-27, is dramatically lowered in transformed cells, with consequent effects on the expression of miR-27 target genes. Transient knockdown and ectopic expression of HSUR 1 demonstrate that it directs degradation of mature miR-27 in a sequence-specific and binding-dependent manner. This viral strategy illustrates use of a ncRNA to manipulate host-cell gene expression via the miRNA pathway.

EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency.

Epstein–Barr virus (EBV) controls gene expression to transform human B cells and maintain viral latency. High‐throughput sequencing and crosslinking immunoprecipitation (HITS‐CLIP) identified mRNA targets of 44 EBV and 310 human microRNAs (miRNAs) in Jijoye (Latency III) EBV‐transformed B cells. While 25% of total cellular miRNAs are viral, only three viral mRNAs, all latent transcripts, are targeted. Thus, miRNAs do not control the latent/lytic switch by targeting EBV lytic genes. Unexpectedly, 90% of the 1664 human 3′‐untranslated regions targeted by the 12 most abundant EBV miRNAs are also targeted by human miRNAs via distinct binding sites. Half of these are targets of the oncogenic miR‐17∼92 miRNA cluster and associated families, including mRNAs that regulate transcription, apoptosis, Wnt signalling, and the cell cycle. Reporter assays confirmed the functionality of several EBV and miR‐17 family miRNA‐binding sites in EBV latent membrane protein 1 (LMP1), EBV BHRF1, and host CAPRIN2 mRNAs. Our extensive list of EBV and human miRNA targets implicates miRNAs in the control of EBV latency and illuminates viral miRNA function in general.

Formation of triple-helical structures by the 3′-end sequences of MALAT1 and MENβ noncoding RNAs

Stability of the long noncoding-polyadenylated nuclear (PAN) RNA from Kaposis sarcoma-associated herpesvirus is conferred by an expression and nuclear retention element (ENE). The ENE protects PAN RNA from a rapid deadenylation-dependent decay pathway via formation of a triple helix between the U-rich internal loop of the ENE and the 3′-poly(A) tail. Because viruses borrow molecular mechanisms from their hosts, we searched highly abundant human long-noncoding RNAs and identified putative ENE-like structures in metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) RNAs. Unlike the PAN ENE, the U-rich internal loops of both predicted cellular ENEs are interrupted by G and C nucleotides and reside upstream of genomically encoded A-rich tracts. We confirmed the ability of MALAT1 and MENβ sequences containing the predicted ENE and A-rich tract to increase the levels of an intronless β-globin reporter RNA. UV thermal denaturation profiles at different pH values support formation of a triple-helical structure composed of multiple U•A-U base triples and a single C•G-C base triple. Additional analyses of the MALAT1 ENE revealed that robust stabilization activity requires an intact triple helix, strong stems at the duplex-triplex junctions, a G-C base pair flanking the triplex to mediate potential A-minor interactions, and the 3′-terminal A of the A-rich tract to form a blunt-ended triplex lacking unpaired nucleotides at the duplex-triplex junction. These examples of triple-helical, ENE-like structures in cellular noncoding RNAs, are unique.

EBV Noncoding RNA Binds Nascent RNA to Drive Host PAX5 to Viral DNA

EBER2 is an abundant nuclear noncoding RNA expressed by the Epstein-Barr virus (EBV). Probing its possible chromatin localization by CHART revealed EBER2s presence at the terminal repeats (TRs) of the latent EBV genome, overlapping previously identified binding sites for the B cell transcription factor PAX5. EBER2 interacts with PAX5 and is required for the localization of PAX5 to the TRs. EBER2 knockdown phenocopies PAX5 depletion in upregulating the expression of LMP2A/B and LMP1, genes nearest the TRs. Knockdown of EBER2 also decreases EBV lytic replication, underscoring the essential role of the TRs in viral replication. Recruitment of the EBER2-PAX5 complex is mediated by base-pairing between EBER2 and nascent transcripts from the TR locus. The interaction is evolutionarily conserved in the related primate herpesvirus CeHV15 despite great sequence divergence. Using base-pairing with nascent RNA to guide an interacting transcription factor to its DNA target site is a previously undescribed function for a trans-acting noncoding RNA.

Conserved motifs in both CPSF73 and CPSF100 are required to assemble the active endonuclease for histone mRNA 3′-end maturation

In eukaryotes, the process of messenger RNA 3′‐end formation involves endonucleolytic cleavage of the transcript followed by synthesis of the poly(A) tail. The complex machinery involved in this maturation process contains two proteins of the metallo‐β‐lactamase (MBL) superfamily, the 73 and 100 kDa subunits of the cleavage and polyadenylation specificity factor (CPSF). By using an in vitro system to assess point mutations in these two mammalian proteins, we found that conserved residues from the MBL motifs of both polypeptides are required for assembly of the endonuclease activity that cleaves histone pre‐mRNAs. This indicates that CPSF73 and CPSF100 act together in the process of maturation of eukaryotic pre‐messenger RNAs, similar to other members of the MBL family, RNases Z and J, which function as homodimers.

Association of Argonaute proteins and microRNAs can occur after cell lysis.

MicroRNA (miRNA) target identification is a challenging but important endeavor. Global analyses of the direct mRNA targets of miRNAs have relied heavily upon immunopurification techniques, wherein a core protein component of the miRNA-protein complex, Argonaute (Ago), is immunoprecipitated to isolate associated RNAs. This approach involves the assumption that the selected RNAs were bound to the Ago protein in vivo and that the methodology did not significantly perturb endogenous interactions or produce novel interaction artifacts. To test whether RNAs that coimmunoprecipitate with human Ago were bound in vivo or could associate post-cell lysis, we used an experimental approach that distinguishes between these two origins of interaction. We show that a transfected miRNA mimic, but not a plasmid-expressed miRNA, can interact with human Ago proteins post-lysis. Our results have important implications for the design of miRNP immunoprecipitation experiments.

Widespread Inducible Transcription Downstream of Human Genes

Pervasive transcription of the human genome generates RNAs whose mode of formation and functions are largely uncharacterized. Here, we combine RNA-seq with detailed mechanistic studies to describe a transcript type derived from protein-coding genes. The resulting RNAs, which we call DoGs for downstream of gene containing transcripts, possess long non-coding regions (often >45 kb) and remain chromatin bound. DoGs are inducible by osmotic stress through an IP3 receptor signaling-dependent pathway, indicating active regulation. DoG levels are increased by decreased termination of the upstream transcript, a previously undescribed mechanism for rapid transcript induction. Relative depletion of polyA signals in DoG regions correlates with increased levels of DoGs after osmotic stress. We detect DoG transcription in several human cell lines and provide evidence for thousands of DoGs genome wide.

EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression

Significance In recent years, next-generation sequencing has facilitated the discovery of thousands of nonprotein-coding RNAs (ncRNAs). Some of these ncRNAs have been shown to interact with transcription regulators and may thus form a new class of ncRNAs that function in controlling gene expression. Identifying proteins that interact with this class of ncRNAs will be instrumental in elucidating their poorly understood molecular mode of action. The ncRNA EBV-encoded RNA 2 (EBER2) expressed by the oncogenic Epstein–Barr virus interacts with the cellular transcription factor paired box protein 5 (PAX5) to impact viral gene expression. In this study, we identified additional functionally important protein components of the EBER2–PAX5 complex. Our findings pave the way for investigating whether other transcription-regulating ncRNAs use the same protein components to exert their function. Epstein–Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2–PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA–protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2.

Comparative analysis reveals genomic features of stress-induced transcriptional readthrough

Significance Cells and organisms live in constantly changing environments. Therefore, cells have evolved complex mechanisms to cope with physiological and environmental stresses. Many of these mechanisms involve transcriptional responses facilitating survival and adaptation. Recent evidence documents extensive transcriptional readthrough beyond annotated gene ends in response to stress, but the role and regulation of these downstream of gene-containing transcripts (DoGs) remain elusive. Here we report that induction of transcriptional readthrough is a hallmark of the mammalian stress response. We explore its causes and consequences in a genome-wide fashion, identifying thousands of readthrough transcripts that are induced in three different stress conditions. Our results suggest potential roles for this class of transcripts in the maintenance of open chromatin under stress. Transcription is a highly regulated process, and stress-induced changes in gene transcription have been shown to play a major role in stress responses and adaptation. Genome-wide studies reveal prevalent transcription beyond known protein-coding gene loci, generating a variety of RNA classes, most of unknown function. One such class, termed downstream of gene-containing transcripts (DoGs), was reported to result from transcriptional readthrough upon osmotic stress in human cells. However, how widespread the readthrough phenomenon is, and what its causes and consequences are, remain elusive. Here we present a genome-wide mapping of transcriptional readthrough, using nuclear RNA-Seq, comparing heat shock, osmotic stress, and oxidative stress in NIH 3T3 mouse fibroblast cells. We observe massive induction of transcriptional readthrough, both in levels and length, under all stress conditions, with significant, yet not complete, overlap of readthrough-induced loci between different conditions. Importantly, our analyses suggest that stress-induced transcriptional readthrough is not a random failure process, but is rather differentially induced across different conditions. We explore potential regulators and find a role for HSF1 in the induction of a subset of heat shock-induced readthrough transcripts. Analysis of public datasets detected increases in polymerase II occupancy in DoG regions after heat shock, supporting our findings. Interestingly, DoGs tend to be produced in the vicinity of neighboring genes, leading to a marked increase in their antisense-generating potential. Finally, we examine genomic features of readthrough transcription and observe a unique chromatin signature typical of DoG-producing regions, suggesting that readthrough transcription is associated with the maintenance of an open chromatin state.