Joan A. Steitz

Switching from repression to activation: microRNAs can up-regulate translation.

AU-rich elements (AREs) and microRNA target sites are conserved sequences in messenger RNA (mRNA) 3′ untranslated regions (3′UTRs) that control gene expression posttranscriptionally. Upon cell cycle arrest, the ARE in tumor necrosis factor–α (TNFα) mRNA is transformed into a translation activation signal, recruiting Argonaute (AGO) and fragile X mental retardation–related protein 1 (FXR1), factors associated with micro-ribonucleoproteins (microRNPs). We show that human microRNA miR369-3 directs association of these proteins with the AREs to activate translation. Furthermore, we document that two well-studied microRNAs—Let-7 and the synthetic microRNA miRcxcr4—likewise induce translation up-regulation of target mRNAs on cell cycle arrest, yet they repress translation in proliferating cells. Thus, activation is a common function of microRNPs on cell cycle arrest. We propose that translation regulation by microRNPs oscillates between repression and activation during the cell cycle.

HuR and mRNA stability

Abstract. An important mechanism of posttranscriptional gene regulation in mammalian cells is the rapid degradation of messenger RNAs (mRNAs) signaled by AU-rich elements (AREs) in their 3′ untranslated regions. HuR, a ubiquitously expressed member of the Hu family of RNA-binding proteins related to Drosophila ELAV, selectively binds AREs and stabilizes ARE-containing mRNAs when overexpressed in cultured cells. This review discusses mRNA decay as a general form of gene regulation, decay signaled by AREs, and the role of HuR and its Hu-family relatives in antagonizing this mRNA degradation pathway. The influence of newly identified protein ligands to HuR on HuR function in both normal and stressed cells may explain how ARE-mediated mRNA decay is regulated in response to environmental change.

Overexpression of HuR, a nuclear–cytoplasmic shuttling protein, increases the in vivo stability of ARE‐containing mRNAs

The messenger RNAs of many proto‐oncogenes, cytokines and lymphokines are targeted for rapid degradation through AU‐rich elements (AREs) located in their 3′ untranslated regions (UTRs). HuR, a ubiquitously expressed member of the Elav family of RNA binding proteins, exhibits specific affinities for ARE‐containing RNA sequences in vitro which correlate with their in vivo decay rates, thereby implicating HuR in the ARE‐mediated degradation pathway. We have transiently transfected HuR into mouse L929 cells and observed that overexpression of HuR enhances the stability of β‐globin reporter mRNAs containing either class I or class II AREs. The increase in mRNA stability parallels the level of HuR overexpression, establishing an in vivo role for HuR in mRNA decay. Furthermore, overexpression of HuR deletion mutants lacking RNA recognition motif 3 (RRM 3) does not exert a stabilizing effect, indicating that RRM 3 is important for HuR function. We have also developed polyclonal anti‐HuR antibodies. Immunofluorescent staining of HeLa and L929 cells using affinity‐purified anti‐HuR antibody shows that both endogenous and overexpressed HuR proteins are localized in the nucleus. By forming HeLa–L929 cell heterokaryons, we demonstrate that HuR shuttles between the nucleus and cytoplasm. Thus, HuR may initially bind to ARE‐containing mRNAs in the nucleus and provide protection during and after their export to the cytoplasmic compartment.

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.

SR Splicing Factors Serve as Adapter Proteins for TAP-Dependent mRNA Export.

The only mammalian RNA binding adapter proteins known to partner with TAP/NXF1, the primary receptor for general mRNA export, are members of the REF family. We demonstrate that at least three shuttling SR (serine/arginine-rich) proteins interact with the same domain of TAP/NXF1 that binds REFs. Included are 9G8 and SRp20, previously shown to promote the export of intronless RNAs. A peptide derived from the N terminus of 9G8 inhibits the binding of both REF and SR proteins to TAP/NXF1 in vitro, and this finding argues for competitive interactions. In Xenopus oocytes, the N terminus of 9G8 exhibits a dominant-negative effect on mRNA export from the nucleus, while addition of excess TAP/NXF1 overcomes this inhibition. Thus, multiple adapters including SR proteins most likely cooperate to recruit multiple copies of TAP/NXF1 for efficient mRNA export.

Sno Storm in the Nucleolus: New Roles for Myriad Small RNPs

these snoRNAs. Yet, as more snoRNAs were discovBiologists have known for decades that the nucleolus ered, not all exhibited the hallmarks of U3, U8, and U13: is the compartment of the eukaryotic cell most densely some possessed an unmodified terminal 59 monophospacked with RNA. But few would have guessed that, in phate instead of the TMG cap and some lacked the addition to the ribosomal RNAs (rRNAs) at various characteristic box C and D sequences (Table 1). Progstages of maturation, the nucleolus contains a multitude ress in assigning functions was made by deletion/rescue of discrete small RNA molecules. These small nucleolar experiments in yeast and in Xenopus oocytes. Certain RNAs, or snoRNAs, are responsible not only for orchessnoRNAs were found to be essential for growth (U3, trating the cleavage events that cut the long pre-rRNA U14, snR10 [temperature sensitive], and snR30) in yeast into 18S, 5.8S, and 28S molecules, but also for adding and/or for specific cleavage steps in pre-rRNA profinishing touches to rRNAs as they assemble into the cessing (U3, U8, and U22) in Xenopus. Curiously, deleultimate products of the nucleolus, the ribosomal subtion of other yeast snoRNA genes had no detectable units. These finishing touches include remodeling of cereffect on growth rate or rRNA maturation (reviewed by tain rRNA uridines into pseudouridines and tagging of Maxwell and Fournier, 1995). numerous ribose moieties with methyl groups. The A completely unanticipated mode of biogenesis for amazing recent realization is that each of these modifithe particularly small (60–90 nucleotides) and less abuncations is directed by its own specific snoRNA that is dant (z104 copies per cell) members of the vertebrate packaged (as are all small nuclear RNAs) into a ribobox C/D snoRNA family then emerged (reviewed by nucleoprotein (snoRNP) particle. Maxwell and Fournier, 1995). Rather than being tranSuggesting some important function, methylated and scribed from theirown genes, these snoRNAs are intronpseudouridylated residues are confined to the most encoded. Liberated by exonucleolytic processing of exhighly conserved portions of rRNA sequences and are cised introns, these stable intronic fragments possess absent from the discarded regions of pre-rRNA. Verte59 monophosphates instead of TMG caps. SnoRNA host brate rRNAs contain approximately 100 methylated suggenes most commonly specify proteins involved in ars, 95 pseudourdines, and 10 methylated bases (Matranslation or ribosome biogenesis; some prominent exden, 1990), whereas yeast rRNAs exhibit about half as amples are ribosomal proteins, nucleolin, and translamany modifications. Eubacteria display a larger number tion factors. A surprising exception is the U22 host gene of elaborate base modifications and only a few sugar(UHG), whose spliced exons do not appear to produce methylated and pseudouridylated residues. The true a protein product; from its introns, however, are released purpose of these myriad modifications has been unclear U22 and seven other fibrillarin-associated snoRNAs no though it has been postulated that rRNA modifications longer than 85 nucleotides (Tycowski et al., 1996a). contribute in subtle ways to ribosome function. Ribose Characteristic of the shorter box C/D snoRNAs is the methylation may stabilize rRNA by increasing hydrophopresence of extensive sequence complementarity (rangbic interaction surfaces; isomerization of uridine into ing from 10 to 21 nucleotides) to highly conserved repseudouridine creates the potential for an additional gions of rRNA. Thus, a snoRNA:rRNA duplex could theohydrogen bond at the N-1 position, which may contribretically form upstream of either box D or an internal ute to rRNA folding. Also mysterious has been the mechbox D sequence, termed D9 (Figure 1A). Following the anism by which specific rRNA sites are selected for discovery of UHG, a number of approaches—creation modification since no obvious signals, either consensus of a human intron-encoded RNA library (Kiss-Laszlo et sequences or local secondary structures, are apparent. al., 1996), database searches for species with boxes C The answer, we now know, lies in simple base pairing. and D as well as complementarity to rRNA (Nicoloso et A snoRNA exhibits extensive complementarity to the al., 1996), and electrophoretic isolation from HeLa cells rRNA sequence flanking the nucleotide to be modified (Tycowski et al., 1996b)—were employed to identify over and directs, according to its class, either sugar methyla30 new examples of the so-called antisense snoRNAs.

Splicing double: insights from the second spliceosome

Almost 20 years after the discovery of introns and RNA splicing, a second spliceosome was uncovered. Although this new spliceosome is structurally and functionally analogous to the well-characterized major-class splicing apparatus, it mediates the excision of a minor class of evolutionarily conserved introns that have non-canonical consensus sequences. This unanticipated diversity in the splicing machinery is refining both the mechanistic understanding and evolutionary models of RNA splicing.

Splicing Factors SRp20 and 9G8 Promote the Nucleocytoplasmic Export of mRNA

We have uncovered a novel function for two members of the SR protein family in mRNA export. Using UV cross-linking, transient transfection, and Xenopus oocyte microinjection, we find that the nucleocytoplasmic shuttling proteins SRp20 and 9G8 interact specifically with a 22-nt RNA element from the histone H2a gene to promote the export of intronless RNAs in both mammalian cells and Xenopus oocytes. Antibodies to SRp20 or 9G8 eliminate RNA binding and significantly inhibit the export of RNAs carrying the element from oocyte nuclei. Our observation that SRp20 and 9G8 can be UV cross-linked to polyadenylated RNA in both the nucleus and cytoplasm of HeLa cells suggests a more general role for these SR proteins in mRNA export.

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.

Identification of HuR as a protein implicated in AUUUA-mediated mRNA decay

Expression of many proto‐oncogenes, cytokines and lymphokines is regulated by targeting their messenger RNAs for rapid degradation. Essential signals for this control are AU‐rich elements (AREs) in the 3′ untranslated region (UTR) of these messages. The ARE is loosely defined as the five‐nucleotide sequence AUUUA embedded in a uracil‐rich region. A trans‐acting factor, presumably a protein, binds the ARE and initiates recognition by the destabilization machinery. Numerous candidate ARE‐binding proteins have been proposed. We show that a 32 kDa protein in HeLa nuclear extracts characterized previously has RNA‐binding specificity that correlates with the activity of an ARE in directing mRNA decay. Purification and subsequent analyses demonstrate that this 32 kDa protein is identical to a recently identified member of the Elav‐like gene family (ELG) called HuR. The in vitro binding selectivity of HuR is indicative of an ARE sequences ability to destabilize a mRNA in vivo, suggesting a critical role for HuR in the regulation of mRNA degradation.