The RasGAP-associated endoribonuclease G3BP assembles stress granules

Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents, and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149, whose dephosphorylation is induced by arsenite treatment. Critically, a phosphomimetic mutant (S149E) fails to oligomerize and to assemble SGs, whereas a nonphosphorylatable G3BP mutant (S149A) does both. These results suggest that G3BP is an effector of SG assembly, and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.     

Anomalous RNA substrates for mammalian tRNA 3' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. The enzyme can also recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA. To investigate the interaction between 3' tRNase and substrates, we tested various anomalous pre-tRNA-like complexes for cleavage by pig 3' tRNase. We examined how base mismatches in the acceptor stem affect 3' tRNase cleavage of RNA complexes, and found that even one base mismatch in the acceptor stem drastically reduces the cleavage efficiency. Mammalian 3' tRNase was able to recognize complexes between target RNAs and 5'-half tDNAs, and cleave the target RNAs, although inefficiently, whereas the enzyme had no activity to cleave phosphodiester bonds of DNA. A relatively long RNA target, the Escherichia coli chloramphenicol acetyltransferase (CAT) mRNA, was cleaved by 3' tRNase in the presence of appropriate 5'-half tRNAs. We also demonstrated that an RNA complex of lin-4 and lin-14 from Caenorhabditis elegans can be recognized and cleaved by pig 3' tRNase.     

RNA heptamers that direct RNA cleavage by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can recognize and cleave any target RNA that forms a precursor tRNA-like complex with another RNA. Various sets of RNA molecules were tested to identify the smallest RNA that can direct target RNA cleavage by 3' tRNase. A 3' half tRNAArgwas cleaved efficiently by 3' tRNase in the presence of small 5' half tRNAArgvariants, the D stem-loop region of which was partially deleted. Remarkably, 3' tRNase also cleaved the 3' half tRNAArgin the presence of a 7 nt 5' tRNAArg composed only of the acceptor stem region with a catalytic efficiency comparable with that of cleavage directed by an intact 5' half tRNAArg. The catalytic efficiency of cleavage directed by the heptamer decreased as the stability of the T stem-loop structures of 3' half tRNAArg variants decreased. No heptamer-directed cleavage of a 3' half tRNAArg without T stem base pairs was detected. A heptamer also directed cleavage of an HIV-1 RNA containing a stable hairpin structure. These findings suggest that in the presence of an RNA heptamer, 3' tRNase can discriminate and eliminate target RNAs that possess a stable hairpin adjacent to the heptamer binding sequence from a large complex RNA pool.     

RNA recognition and cleavage by the SARS coronavirus endoribonuclease

The emerging disease SARS is caused by a novel coronavirus that encodes several unusual RNA-processing enzymes, including non-structural protein 15 (Nsp15), a hexameric endoribonuclease that preferentially cleaves at uridine residues. How Nsp15 recognizes and cleaves RNA is not well understood and is the subject of this study. Based on the analysis of RNA products separated by denaturing gel electrophoresis, Nsp15 has been reported to cleave both 5' and 3' of the uridine. We used several RNAs, including some with nucleotide analogs, and mass spectrometry to determine that Nsp15 cleaves only 3' of the recognition uridylate, with some cleavage 3' of cytidylate. A highly conserved RNA structure in the 3' non-translated region of the SARS virus was cleaved preferentially at one of the unpaired uridylate bases, demonstrating that both RNA structure and base-pairing can affect cleavage by Nsp15. Several modified RNAs that are not cleaved by Nsp15 can bind Nsp15 as competitive inhibitors. The RNA binding affinity of Nsp15 increased with the content of uridylate in substrate RNA and the co-factor Mn(2+). The hexameric form of Nsp15 was found to bind RNA in solution. A two-dimensional crystal of Nsp15 in complex with RNA showed that at least two RNA molecules could be bound per hexamer. Furthermore, an 8.3 A structure of Nsp15 was developed using cyroelectron microscopy, allowing us to generate a model of the Nsp15-RNA complex.     

Conversion of mammalian tRNA 3' processing endoribonuclease to four-base-recognizing RNA cutters.

The spermidine-dependent, sequence-specific endoribonuclease (RNase 65) activities in mammalian cell extracts require both protein and 3' truncated tRNA, species of which direct their substrate sequence specificity. Computer analysis for searching possible base pairing between substrate RNAs and their corresponding 3' truncated tRNA, suggested a unified model for substrate recognition mechanism, in which a four-nucleotide (nt) sequence in the target tRNAs 1 nt upstream of their cleavage site, base pairs with the 5' terminal 4 nt sequence of their corresponding 3' truncated tRNA. This model was supported by experiments with several RNA substrates containing a substituted nucleotide in the target 4 nt sequence. In this model, the tRNA substrates and their corresponding 3' truncated tRNA form a complex resembling a 5' processed tRNA precursor containing a 3' trailer, suggesting that the protein component of RNase 65 is identical to tRNA 3' processing endoribonuclease (3' tRNase). Actually, 3' tRNase purified from pig liver cleaved the target RNAs at the expected sites only in the presence of their corresponding 3' truncated tRNA. These results show that the 3' tRNase can be converted to 4 nt specific RNA cutters using the 3' truncated tRNAs.     

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.     

Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation

Binding of APOBEC3G to the nucleocapsid (NC) domain of the human immunodeficiency virus (HIV) Gag polyprotein may represent a critical early step in the selective packaging of this antiretroviral factor into HIV virions. Previously, we and others have reported that this interaction is mediated by RNA. Here, we demonstrate that RNA binding by APOBEC3G is key for initiation of APOBEC3G:NC complex formation in vitro. By adding back nucleic acids to purified, RNase-treated APOBEC3G and NC protein preparations in vitro, we demonstrate that complex formation is rescued by short (> or =10 nucleotides) single-stranded RNAs (ssRNAs) containing G residues. In contrast, complex formation is not induced by add-back of short ssRNAs lacking G, by dsRNAs, by ssDNAs, by dsDNAs or by DNA:RNA hybrid molecules. While some highly structured RNA molecules, i.e., tRNAs and rRNAs, failed to rescue APOBEC3G:NC complex formation, other structured RNAs, i.e., human Y RNAs and 7SL RNA, did promote NC binding by APOBEC3G. Together, these results indicate that ternary complex formation requires ssRNA, but suggest this can be presented in the context of an otherwise highly structured RNA molecule. Given previous data arguing that APOBEC3G binds, and edits, ssDNA effectively in vitro, these data may also suggest that APOBEC3G can exist in two different conformational states, with different activities, depending on whether it is bound to ssRNA or ssDNA.     

Microtubule-associated protein 1B: molecular structure, localization, and phosphorylation-dependent expression in developing neurons

Two monoclonal antibodies, 5E6 and 1B6, were raised against microtubule-associated protein 1B (MAP1B), a major component of the neuronal cytoskeleton. 5E6 recognized the entire MAP1B population, while 1B6 detected only phosphorylated forms. Affinity-purified MAP1B appeared as a long, filamentous molecule (186 +/- 38 nm) with a small spherical portion at one end, forming long cross-bridges between microtubules in vitro. These results, together with in vivo data from immunogold methods, demonstrate that MAP1B is a component of cross-bridges between microtubules in neurons. By immunohistochemical analysis, phosphorylated forms were shown to exist mainly in axons, whereas unphosphorylated forms were limited to cell bodies and dendrites. Phosphorylated MAP1B was quite abundant in developing axons, suggesting its essential role in axonal elongation.     

RNA localization and transport

RNA localization serves numerous purposes from controlling development and differentiation to supporting the physiological activities of cells and organisms. After a brief introduction into the history of the study of mRNA localization I will focus on animal systems, describing in which cellular compartments and in which cell types mRNA localization was observed and studied. In recent years numerous novel localization patterns have been described, and countless mRNAs have been documented to accumulate in specific subcellular compartments. These fascinating revelations prompted speculations about the purpose of localizing all these mRNAs. In recent years experimental evidence for an unexpected variety of different functions has started to emerge. Aside from focusing on the functional aspects, I will discuss various ways of localizing mRNAs with a focus on the mechanism of active and directed transport on cytoskeletal tracks. Structural studies combined with imaging of transport and biochemical studies have contributed to the enormous recent progress, particularly in understanding how dynein/dynactin/BicD (DDB) dependent transport on microtubules works. This transport process actively localizes diverse cargo in similar ways to the minus end of microtubules and, at least in flies, also individual mRNA molecules. A sophisticated mechanism ensures that cargo loading licenses processive transport.     

Soluble endoribonuclease activity from vaccinia virus: specific cleavage of virion-associated high-molecular-weight RNA.

A soluble endoribonuclease activity was extracted from purified vaccinia virus cores by treatment with sodium-deoxycholate and dithiothreitol. The soluble enzyme readily cleaved purified virion-associated high-molecular-weight RNA to limit-sized fragments sedimenting at 8 to 12S. Purified virion-released 8 to 12S polyadenylated mRNA was not degraded by the enzyme extract. The soluble endoribonuclease did not require the presence of ribonucleoside triphosphates for activity.     

RNA quality control: degradation of defective transfer RNA

The distinction between stable (tRNA and rRNA) and unstable (mRNA) RNA has been considered an important feature of bacterial RNA metabolism. One factor thought to contribute to the difference between these RNA populations is polyadenylation, which promotes degradation of unstable RNA. However, the recent discovery that polyadenylation also occurs on stable RNA led us to examine whether poly(A) might serve as a signal for eliminating defective stable RNAs, and thus play a role in RNA quality control. Here we show that a readily denaturable, mutant tRNA(Trp) does not accumulate to normal levels in Escherichia coli because its precursor is rapidly degraded. Degradation is largely dependent on polyadenylation of the precursor by poly(A) polymerase and on its removal by polynucleotide phosphorylase. Thus, in the absence of these two enzymes large amounts of tRNA(Trp) precursor accumulate. We propose that defective stable RNA precursors that are poorly converted to their mature forms may be polyadenylated and subsequently degraded. These data indicate that quality control of stable RNA metabolism in many ways resembles normal turnover of unstable RNA.     

RNA localization: visualization in real-time

RNA localization during development is required for proper sorting of developmental determinants. Direct visualization of endogenous transcribed RNA is now possible and should provide new insights into how this process occurs.     

RNA and memory: from feeding to localization

Ideas about the role of RNA in learning and memory have a checkered past. A new study in Drosophila, synthesizing classical forward genetics with DNA microarrays, brings us closer to seeing that role clearly.     

Mitochondrial polyadenylic acid-containing RNA: localization and characterization

The RNA from the mitochondrial fraction of animal cells contains a polyadenylic acid sequence, approximately 55 nucleotides in length, which migrates at about 4 S in gel electrophoresis and which is attached to high molecular weight RNA. The experiments reported here indicate that: (a) the 4 S poly(A) sequence is found only in the mitochondrial fraction; (b) the RNA containing 4 S poly(A) is located within structures (presumably mitochondria) which protect it from pancreatic ribonuclease; (c) no RNA containing the longer poly(A) of nuclear origin appears to be located in mitochondria; (d) the 4 S poly(A), but not the longer poly(A), is attached to RNA which hybridizes to mitochondrial DNA; and (e) this poly(A) sequence is located at the 3′ end of the RNA molecule.The poly(A)-containing RNA can be isolated by affinity to oligodeoxyribothymidylic acid cellulose and resolved into approximately eight distinct species by acrylamide gel electrophoresis. These may correspond to individual mitochondrial messenger RNA molecules.