The SRP9/14 subunit of the human signal recognition particle binds to a variety of Alu-like RNAs and with higher affinity than its mouse homolog.

The heterodimeric subunit, SRP9/14, of the signal recognition particle (SRP) has previously been found to bind to scAlu and scB1 RNAs in vitro and to exist in large excess over SRP in anthropoid cells. Here we show that human and mouse SRP9/14 bind with high affinities to other Alu-like RNAs of different evolutionary ages including the neuron-specific BC200 RNA. The relative dissociation constants of the different RNA-protein complexes are inversely proportional to the evolutionary distance between the Alu RNA species and 7SL RNA. In addition, the human SRP9/14 binds with higher affinity than mouse SRP9/14 to all RNAs analyzed and this difference is not explained by the additional C-terminal domain present in the anthropoid SRP14. The conservation of high affinity interactions between SRP9/14 and Alu-like RNAs strongly indicates that these Alu-like RNPs exist in vivo and that they have cellular functions. The observation that human SRP9/14 binds better than its mouse counterpart to distantly related Alu RNAs, such as recently transposed elements, suggests that the anthropoid-specific excess of SRP9/14 may have a role in controlling Alu amplification rather than in compensating a defect in SRP assembly and functions.     

Alu RNP and Alu RNA regulate translation initiation in vitro

Alu elements are the most abundant repetitive elements in the human genome; they emerged from the signal recognition particle RNA gene and are composed of two related but distinct monomers (left and right arms). Alu RNAs transcribed from these elements are present at low levels at normal cell growth but various stress conditions increase their abundance. Alu RNAs are known to bind the cognate proteins SRP9/14. We purified synthetic Alu RNP, composed of Alu RNA in complex with SRP9/14, and investigated the effects of Alu RNPs and naked Alu RNA on protein translation. We found that the dimeric Alu RNP and the monomeric left and right Alu RNPs have a general dose-dependent inhibitory effect on protein translation. In the absence of SRP9/14, Alu RNA has a stimulatory effect on all reporter mRNAs. The unstable structure of sRight RNA suggests that the differential activities of Alu RNP and Alu RNA may be explained by conformational changes in the RNA. We demonstrate that Alu RNPs and Alu RNAs do not stably associate with ribosomes during translation and, based on the analysis of polysome profiles and synchronized translation, we show that Alu RNP and Alu RNA regulate translation at the level of initiation.     

Direct binding of the Alu binding protein dimer SRP9/14 to 40S ribosomal subunits promotes stress granule formation and is regulated by Alu RNA

Stress granules (SGs) are formed in response to stress, contain mRNAs, 40S ribosomal subunits, initiation factors, RNA-binding and signaling proteins, and promote cell survival. Our study describes a novel function of the protein heterodimer SRP9/14 and Alu RNA in SG formation and disassembly. In human cells, SRP9/14 exists assembled into SRP, bound to Alu RNA and as a free protein. SRP9/14, but not SRP, localizes to SGs following arsenite or hippuristanol treatment. Depletion of the protein decreases SG size and the number of SG-positive cells. Localization and function of SRP9/14 in SGs depend primarily on its ability to bind directly to the 40S subunit. Binding of SRP9/14 to 40S and Alu RNA is mutually exclusive indicating that the protein alone is bound to 40S in SGs and that Alu RNA might competitively regulate 40S binding. Indeed, by changing the effective Alu RNA concentration in the cell or by expressing an Alu RNA binding-defective protein we were able to influence SG formation and disassembly. Our findings suggest a model in which SRP9/14 binding to 40S promotes SG formation whereas the increase in cytoplasmic Alu RNA following stress promotes disassembly of SGs by disengaging SRP9/14 from 40S.     

A highly conserved nucleotide in the Alu domain of SRP RNA mediates translation arrest through high affinity binding to SRP9/14.

Binding of the signal recognition particle (SRP) to signal sequences during translation leads to an inhibition of polypeptide elongation known as translation arrest. The arrest activity is mediated by a discrete domain comprised of the Alu portion of SRP RNA and a 9 and 14 kDa polypeptide heterodimer (SRP9/14). Although very few nucleotides in SRP RNA are conserved throughout evolution, the remarkable conservation of G24, which resides in the region of SRP9/14 interaction, suggests that it is essential for translation arrest. To understand the functional significance of the G24 residue, we made single base substitutions in SRP RNA at this position and analyzed the ability of the mutants to bind SRP9/14 and to reconstitute functional SRPs. Mutation of G24 to C reduced binding to SRP9/14 by at least 50-fold, whereas mutation to A and U reduced binding approximately 2- and 5-fold respectively. The mutant RNAs could nevertheless assemble into SRPs at high subunit concentrations. SRPs reconstituted with mutant RNAs were not significantly defective in translation arrest assays, indicating that the conserved guanosine does not interact directly with the translational machinery. Taken together, these results demonstrate that G24 plays an important role in the translation arrest function of SRP by mediating high affinity binding of SRP9/14.     

Plant virus RNAs. Coordinated recruitment of conserved host functions by (+) ssRNA viruses during early infection events

Positive-sense single-stranded RNA viruses have developed strategies to exploit cellular resources at the expense of host mRNAs. The genomes of these viruses display a variety of structures at their 5' and 3' ends that differentiate them from cellular mRNAs. Despite this structural diversity, viral RNAs are still circularized by juxtaposition of their 5' and 3' ends, similar to the process used by cellular mRNAs. Also reminiscent of the mechanisms used by host mRNAs, translation of viral RNAs involves the recruitment of translation initiation factors. However, the roles played by these factors likely differ from those played by cellular mRNAs. In keeping with the general parsimony typical of RNA viruses, these host factors also participate in viral RNA replication. However, the dual use of host factors requires that viral RNA template utilization be regulated to avoid conflict between replication and translation. The molecular composition of the large ribonucleoprotein complexes that form the viral RNA replication and translation machineries likely evolves over the course of infection to allow for switching template use from translation to replication.     

Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation

Ribosome recruitment to eukaryotic mRNAs is generally thought to occur by a scanning mechanism, whereby the 40S ribosomal subunit binds in the vicinity of the 5'cap structure of the mRNA and scans until an AUG codon is encountered in an appropriate sequence context. Study of the picornaviruses allowed the characterization of an alternative mechanism of translation initiation. Picornaviruses can initiate translation via an internal ribosome entry segment (IRES), an RNA structure that directly recruits the 40S ribosomal subunits in a cap and 5' end independent fashion. Since its discovery, the notion of IRESs has extended to a number of different virus families and cellular RNAs. This review summarizes features of both cap-dependent and IRES-dependent mechanisms of translation initiation and discusses the role of cis-acting elements, which include the 5' cap, the 5'-untranslated region (UTR) and the poly(A) tail as well as the possible roles of IRESs as part of a cellular stress response mechanism and in the virus replication cycle.     

The Alu domain homolog of the yeast signal recognition particle consists of an Srp14p homodimer and a yeast-specific RNA structure

The mammalian Alu domain of the signal recognition particle (SRP) consists of a heterodimeric protein SRP9/14 and the Alu portion of 7SL RNA and comprises the elongation arrest function of the particle. To define the domain in Saccharomyces cerevisiae SRP that is homologous to the mammalian Alu domain [Alu domain homolog in yeast (Adhy)], we examined the assembly of a yeast protein homologous to mammalian SRP14 (Srp14p) and scR1 RNA. Srp14p binds as a homodimeric complex to the 5' sequences of scR1 RNA. Its minimal binding site consists of 99 nt. (Adhy RNA), comprising a short hairpin structure followed by an extended stem. As in mammalian SRP9/14, the motif UGUAAU present in most SRP RNAs is part of the Srp14p binding sites as shown by footprint and mutagenesis studies. In addition, certain basic amino acid residues conserved between mammalian SRP14 and Srp14p are essential for RNA binding in both proteins. These findings confirm the common ancestry of the yeast and the mammalian components and indicate that Srp14p together with Adhy RNA represents the Alu domain homolog in yeast SRP that may comprise its elongation arrest function. Despite the similarities, Srp14p selectively recognizes only scR1 RNA, revealing substantial changes in RNA-protein recognition as well as in the overall structure of the complex. The alignment of the three yeast SRP RNAs known to date suggests a common structure for the putative elongation arrest domain of all three organisms.     

Virion packaging determinants and reverse transcription of SRP RNA in HIV-1 particles

Diverse retroviruses have been shown to package host SRP (7SL) RNA. However, little is known about the viral determinants of 7SL RNA packaging. Here we demonstrate that 7SL RNA is more selectively packaged into HIV-1 virions than are other abundant Pol-III-transcribed RNAs, including Y RNAs, 7SK RNA, U6 snRNA and cellular mRNAs. The majority of the virion-packaged 7SL RNAs were associated with the viral core structures and could be reverse-transcribed in HIV-1 virions and in virus-infected cells. Viral Pol proteins influenced tRNAlys,3 packaging but had little influence on virion packaging of 7SL RNA. The N-terminal basic region and the basic linker region of HIV-1 NCp7 were found to be important for efficient 7SL RNA packaging. Although Alu RNAs are derived from 7SL RNA and share the Alu RNA domain with 7SL RNA, the packaging of Alu RNAs was at least 50-fold less efficient than that of 7SL RNA. Thus, 7SL RNAs are selectively packaged into HIV-1 virions through mechanisms distinct from those for viral genomic RNA or primer tRNAlys,3. Virion packaging of both human cytidine deaminase APOBEC3G and cellular 7SL RNA are mapped to the same regions in HIV-1 NC domain.     

The crystal structure of the signal recognition particle Alu RNA binding heterodimer, SRP9/14.

The mammalian signal recognition particle (SRP) is an 11S cytoplasmic ribonucleoprotein that plays an essential role in protein sorting. SRP recognizes the signal sequence of the nascent polypeptide chain emerging from the ribosome, and targets the ribosome-nascent chain-SRP complex to the rough endoplasmic reticulum. The SRP consists of six polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72) and a single 300 nucleotide RNA molecule. SRP9 and SRP14 proteins form a heterodimer that binds to the Alu domain of SRP RNA which is responsible for translation arrest. We report the first crystal structure of a mammalian SRP protein, that of the mouse SRP9/14 heterodimer, determined at 2.5 A resolution. SRP9 and SRP14 are found to be structurally homologous, containing the same alpha-beta-beta-beta-alpha fold. This we designate the Alu binding module (Alu bm), an additional member of the family of small alpha/beta RNA binding domains. The heterodimer has pseudo 2-fold symmetry and is saddle like, comprising a strongly curved six-stranded amphipathic beta-sheet with the four helices packed on the convex side and the exposed concave surface being lined with positively charged residues.     

Assembly of the Alu domain of the signal recognition particle (SRP): dimerization of the two protein components is required for efficient binding to SRP RNA.

The signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, plays an essential role in targeting secretory proteins to the rough endoplasmic reticulum membrane. In addition to the targeting function, SRP contains an elongation arrest or pausing function. This function is carried out by the Alu domain, which consists of two proteins, SRP9 and SRP14, and the portion of SRP (7SL) RNA which is homologous to the Alu family of repetitive sequences. To study the assembly pathway of the components in the Alu domain, we have isolated a cDNA clone of SRP9, in addition to a previously obtained cDNA clone of SRP14. We show that neither SRP9 nor SRP14 alone interacts specifically with SRP RNA. Rather, the presence of both proteins is required for the formation of a stable RNA-protein complex. Furthermore, heterodimerization of SRP9 and SRP14 occurs in the absence of SRP RNA. Since a partially reconstituted SRP lacking SRP9 and SRP14 [SRP(-9/14)] is deficient in the elongation arrest function, it follows from our results that both proteins are required to assemble a functional domain. In addition, SRP9 and SRP14 synthesized in vitro from synthetic mRNAs derived from their cDNA clones restore elongation arrest activity to SRP(-9/14).     

Minimum internal ribosome entry site required for poliovirus infectivity.

Translation initiation by internal ribosome binding is a recently discovered mechanism of eukaryotic viral and cellular protein synthesis in which ribosome subunits interact with the mRNAs at internal sites in the 5' untranslated RNA sequences and not with the 5' methylguanosine cap structure present at the extreme 5' ends of mRNA molecules. Uncapped poliovirus mRNAs harbor internal ribosome entry sites (IRES) in their long and highly structured 5' noncoding regions. Such IRES sequences are required for viral protein synthesis. In this study, a novel poliovirus was isolated whose genomic RNA contains two gross deletions removing approximately 100 nucleotides from the predicted IRES sequences within the 5' noncoding region. The deletions originated from previously in vivo-selected viral revertants displaying non-temperature-sensitive phenotypes. Each revertant had a different predicted stem-loop structure within the 5' noncoding region of their genomic RNAs deleted. The mutant poliovirus (Se1-5NC-delta DG) described in this study contains both stem-loop deletions in a single RNA genome, thereby creating a minimum IRES. Se1-5NC-delta DG exhibited slow growth and a pinpoint plaque phenotype following infection of HeLa cells, delayed onset of protein synthesis in vivo, and defective initiation during in vitro translation of the mutated poliovirus mRNAs. Interestingly, the peak levels of viral RNA synthesis in cells infected with Se1-5NC-delta DG occurred at slightly later times in infection than those achieved by wild-type poliovirus, but these mutant virus RNAs accumulated in the host cells during the late phases of virus infection. UV cross-linking assays with the 5' noncoding regions of wild-type and mutated RNAs were carried out in cytoplasmic extracts from HeLa cells and neuronal cells and in reticulocyte lysates to identify the cellular factors that interact with the putative IRES elements. The cellular proteins that were cross-linked to the minimum IRES may represent factors playing an essential role in internal translation initiation of poliovirus mRNAs.     

Binding sites of the 9- and 14-kilodalton heterodimeric protein subunit of the signal recognition particle (SRP) are contained exclusively in the Alu domain of SRP RNA and contain a sequence motif that is conserved in evolution.

The mammalian signal recognition particle (SRP) is a small cytoplasmic ribonucleoprotein required for the cotranslational targeting of secretory proteins to the endoplasmic reticulum membrane. The heterodimeric protein subunit SRP9/14 was previously shown to be essential for SRP to cause pausing in the elongation of secretory protein translation. RNase protection and filter binding experiments have shown that binding of SRP9/14 to SRP RNA depends solely on sequences located in a domain of SRP RNA that is strongly homologous to the Alu family of repetitive DNA sequences. In addition, the use of hydroxyl radicals, as RNA-cleaving reagents, has revealed four distinct regions in this domain that are in close contact with SRP9/14. Surprisingly, the nucleotide sequence in one of these contact sites, predicted to be mostly single stranded, was found to be extremely conserved in SRP RNAs of evolutionarily distant organisms ranging from eubacteria and archaebacteria to yeasts and higher eucaryotic cells. This finding suggests that SRP9/14 homologs may also exist in these organisms, where they possibly contribute to the regulation of protein synthesis similar to that observed for mammalian SRP in vitro.     

Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus translation regulation

Alphavirus infection results in the shutoff of host protein synthesis in favor of viral translation. Here, we show that during Semliki Forest virus (SFV) infection, the translation inhibition is largely due to the activation of the cellular stress response via phosphorylation of eukaryotic translation initiation factor 2alpha subunit (eIF2alpha). Infection of mouse embryo fibroblasts (MEFs) expressing a nonphosphorylatable mutant of eIF2alpha does not result in efficient shutoff, despite efficient viral protein production. Furthermore, we show that the SFV translation enhancer element counteracts the translation inhibition imposed by eIF2alpha phosphorylation. In wild-type MEFs, viral infection induces the transient formation of stress granules (SGs) containing the cellular TIA-1/R proteins. These SGs are disassembled in the vicinity of viral RNA replication, synchronously with the switch from cellular to viral gene expression. We propose that phosphorylation of eIF2alpha and the consequent SG assembly is important for shutoff to occur and that the localized SG disassembly and the presence of the enhancer aid the SFV mRNAs to elude general translational arrest.     

Bridging IRES elements in mRNAs to the eukaryotic translation apparatus

IRES elements are highly structured RNA sequences that function to recruit ribosomes for the initiation of translation. In contrast to the canonical cap-binding, ribosome-scanning model, the mechanism of IRES-mediated translation initiation is not well understood. IRES elements, first discovered in viral RNA genomes, were subsequently found in a subset of cellular RNAs as well. Interestingly, these cellular IRES-containing mRNAs appear to play important roles during conditions of cellular stress, development, and disease (e.g., cancer). It has been shown for viral IRESes that some require specific IRES trans-acting factors (ITAFs), while others require few if any additional proteins and can bind ribosomes directly. Current studies are aimed at elucidating the mechanism of IRES-mediated translation initiation and features that may be common or differ greatly among cellular and viral IRESes. This review will explore IRES elements as important RNA structures that function in both cellular and viral RNA translation and the significance of these structures in providing an alternative mechanism of eukaryotic translation initiation.     

Internal ribosome entry sites of viral and cellular RNAs

In recent years mechanism of internal initation of translation in eukaryotic cells commands the attention of molecular biologists in increasing frequency. Ten years ago, experiments with picornaviruses demonstrated the ability of 40S ribosomal subunits to bind to nucleotide sequences localized far from the 5′ ends of RNA molecules, and since then numerous viral and even cellular RNAs were shown to be capable of internal initiation of translation. In the present survey, data on the localization, structure, and functional load of these internal ribosome entry sites (IRES elements) of viral and cellular RNAs, as well as on proteins capable of strong and highly specific binding to IRES elements, are discussed. A conclusion is that a unified model of structure and fuctioning of viral and cellular IRES elements cannot be suggested.     

Identification of a minimal Alu RNA folding domain that specifically binds SRP9/14.

We have identified functionally and analyzed a minimal Alu RNA folding domain that is recognized by SRPphi14-9. Recombinant SRPphi14-9 is a fusion protein containing on a single polypeptide chain the sequences of both the SRP14 and SRP9 proteins that are part of the Alu domain of the signal recognition particle (SRP). SRPphi14-9 has been shown to bind to the 7SL RNA of SRP and it confers elongation arrest activity to reconstituted SRP in vitro. Alu RNA variants with homogeneous 3' ends were produced in vitro using ribozyme technology and tested for specific SRPphi14-9 binding in a quantitative equilibrium competition assay. This enabled identification of an Alu RNA of 86 nt (SA86) that competes efficiently with 7SL RNA for SRPphi14-9 binding, whereas smaller RNAs did not. The secondary structure of SA86 includes two stem-loops that are connected by a highly conserved bulge and, in addition, a part of the central adaptor stem that contains the sequence at the very 3' end of 7SL RNA. Circularly permuted variants of SA86 competed only if the 5' and 3' ends were joined with an extended linker of four nucleotides. SA86 can thus be defined as an autonomous RNA folding unit that does not require its 5' and 3' ends for folding or for specific recognition by SRPphi14-9. These results suggest that Alu RNA identity is determined by a characteristic tertiary structure, which might consist of two flexibly linked domains.     

Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2alpha

Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m(7)G-cap and then moves along the mRNA until an initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5'-untranslated regions, which allow initiation independently of the 5'-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation initiation factor, eIF2alpha, by activating specific kinases: (i) amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER kinase, PERK kinase; and (iii) double-stranded RNA, which activates double-stranded RNA-dependent protein kinase (PKR) by mimicking viral infection. Amino acid starvation and ER stress caused transient phosphorylation of eIF2alpha during the first hour of treatment, whereas double-stranded RNA caused a sustained phosphorylation of eIF2alpha after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the protein kinase Pim-1. In contrast, translation mediated by the IRESs from the cationic amino acid transporter, cat-1, and of the cricket paralysis virus intergenic region, were stimulated 3- to 10-fold by all three treatments. eIF2alpha phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2alpha. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5'-untranslated regions.     

The ATP requirement for initiation of eukaryotic translation varies according to the mRNA species

The requirement for ATP for initiation of eukaryotic mRNA translation was tested using gel-filtered rabbit reticulocyte lysates incubated with labelled Met-tRNAfMet and exogenous RNA templates, and assaying the formation of labelled 80S initiation complexes in the presence of GTP, or labelled 40S initiation complexes in the presence of a non-hydrolysable analogue of GTP. Initiation complex formation on globin mRNA, or on capped viral RNAs such as papaya mosaic virus RNA and tobacco mosaic virus RNA, was strongly stimulated by ATP. In contrast, initiation complex formation on (uncapped) encephalomyocarditis virus RNA was uninfluenced by the presence or absence of ATP, which may be correlated with the recent evidence for scanning-independent internal initiation on this viral RNA. In addition, initiation complex formation on uncapped cowpea mosaic virus RNA and on poly(A,U,G) was only slightly stimulated by ATP, much less than in the case of the capped RNAs. These results suggest that most of the ATP hydrolysed during translation initiation is consumed in cap-dependent processes, probably in unwinding the mRNA, and relatively little in the actual migration or scanning of 40S subunits along the mRNA.     

Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses

A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.