The essential yeast RNA binding protein Np13p is methylated

Arginine methylation is a prevalent modification found in many RNA binding proteins, yet little is known about its functional consequences. Using a monoclonal antibody, 1E4, we have shown that the yeast NPL3 gene product Np13p, an essential RNA binding protein with repeated RGG motifs, is arginine-methylated in vivo. The 1E4 epitope can be generated by incubating recombinant Np13p with partially purified bovine arginine methyltransferase block this reaction. Np13p methylation requires S-adenosyl-L-methionine and also occurs in yeast extracts. An Np13p deletion mutant lacking the RGG domain is not a substrate for methylation, suggesting that the methylation sites lie within the RGG motifs. The discovery of arginine methylation in a genetically tractable organism provides a powerful entrée to understanding the function of this modification, particularly in view of the many roles postulated for Np13p in RNA processing and transport. The recent discovery of phosphorylated serine residues within the RGG domain suggests a hypothesis in which a molecular switch governed by methylation and phosphorylation regulates the biochemical properties of the Np13p RGG domain.     

Symptom attenuation by a normally virulent satellite RNA of turnip crinkle virus is associated with the coat protein open reading frame

Many satellite RNAs (sat-RNAs) can attenuate or intensify the symptoms produced by their helper virus. Sat-RNA C, associated with turnip crinkle virus (TCV), was previously found to intensify the symptoms of TCV on all plants in which TCV produced visible symptoms. However, when the coat protein open reading frame (ORF) of TCV was precisely exchanged with that of cardamine chlorotic fleck virus, sat-RNA C attenuated the moderate symptoms of the chimeric virus when Arabidopsis plants were coinoculated with the chimeric virus. Symptom attenuation was correlated with a reduction in viral RNA levels in inoculated and uninoculated leaves. In protoplasts, the presence of sat-RNA C resulted in a reduction of approximately 70% in the chimeric viral genomic RNA at 44 hr postinoculation, whereas the sat-RNA wa consistently amplified to higher levels by the chimeric virus than by wild-type TCV. TCV with a deletion of the coat protein ORF also resulted in a similar increase in sat-RNA C levels in protoplasts, indicating that the TVC coat protein, or its ORF, downregulates the synthesis of sat-RNA C. These results suggest that the coat protein or its ORF is a viral determinant for symptom modulation by sat-RNA C, and symptom attenuation is at least partly due to inhibition of virus accumulation.     

Craf-1 protein kinase is essential for mouse development

The complete cDNA of the mouse integral membrane protein 2B gene (Itm2b) was determined by sequence analysis of expressed sequence tag (EST) clone L26775 and a clone isolated from a cDNA library of the osteogenic stromal cell line MN7 (Mathieu et al., 1992. Calcif. Tissue Int. 50, 362-371) and by 5' rapid amplification of cDNA ends (RACE). Alignment of different mouse ESTs confirmed the entire sequence. Northern blot analysis of different neonatal and adult mouse tissues showed that Itm2b is ubiquitously expressed. There are three mRNAs with different lengths in neonatal as well as in adult tissues, originating from alternative polyadenylation by usage of one consensus and two additional variant polyadenylation signals. The cDNA sequence of the human Itm2b homolog (ITM2B) was assembled using data from available human ESTs. Both the mouse and the human gene code for a protein of 266 amino acids (aa) that is homologous to a previously described integral membrane protein, Itm2A, of which the expression is restricted to osteo- and chondrogenic tissues. Itm2A and Itm2B belong to a family of type II integral membrane proteins, which contains a third member, Itm2C (Deleersnijder et al., 1996. J. Biol. Chem. 271, 19475-19482). The human ITM2B and mouse Itm2b genes were previously mapped as unknown ESTs to conserved syntenic regions Homo sapiens 13q12-13 and Mus musculus 14.     

Complementation of RNA binding site mutations in MS2 coat protein heterodimers

The coat protein of bacteriophage MS2 functions as a symmetric dimer to bind an asymmetric RNA hairpin. This implies the existence of two equivalent RNA binding sites related to one another by a 2-fold symmetry axis. In this view the symmetric binding site defined by mutations conferring the repressor-defective phenotype is a composite picture of these two asymmetric sites. In order to determine whether the RNA ligand interacts with amino acid residues on both subunits of the dimer and in the hope of constructing a functional map of the RNA binding site, we performed heterodimer complementation experiments. Taking advantage of the physical proximity of their N- and C-termini, the two subunits of the dimer were genetically fused, producing a duplicated coat protein which folds normally and allows the construction of the functional equivalent of obligatory heterodimers containing all possible pairwise combinations of the repressor-defective mutations. The restoration of repressor function in certain heterodimers shows that a single RNA molecule interacts with both subunits of the dimer and allows the construction of a functional map of the binding site.     

The N-terminal region of the luteovirus readthrough domain determines virus binding to Buchnera GroEL and is essential for virus persistence in the aphid

Luteoviruses and the luteovirus-like pea enation mosaic virus (PEMV; genus Enamovirus) are transmitted by aphids in a circulative, nonreplicative manner. Acquired virus particles persist for several weeks in the aphid hemolymph, in which a GroEL homolog, produced by the primary endosymbiont of the aphid, is abundantly present. Six subgroup II luteoviruses and PEMV displayed a specific but differential affinity for Escherichia coli GroEL and GroEL homologs isolated from the endosymbiotic bacteria of both vector and nonvector aphid species. These observations suggest that the basic virus-binding capacity resides in a conserved region of the GroEL molecule, although other GroEL domains may influence the efficiency of binding. Purified luteovirus and enamovirus particles contain a major 22-kDa coat protein (CP) and lesser amounts of an approximately 54-kDa readthrough protein, expressed by translational readthrough of the CP into the adjacent open reading frame. Beet western yellows luteovirus (BWYV) mutants devoid of the readthrough domain (RTD) did not bind to Buchnera GroEL, demonstrating that the RTD (and not the highly conserved CP) contains the determinants for GroEL binding. In vivo studies showed that virions of these BWYV mutants were significantly less persistent in the aphid hemolymph than were virions containing the readthrough protein. These data suggest that the Buchnera GroEL-RTD interaction protects the virus from rapid degradation in the aphid. Sequence comparison analysis of the RTDs of different luteoviruses and PEMV identified conserved residues potentially important in the interaction with Buchnera GroEL.     

RNA-binding protein TIAR is essential for primordial germ cell development

Primordial germ cells (PGCs) give rise to both eggs and sperm via complex maturational processes that require both cell migration and proliferation. However, little is known about the genes controlling gamete formation during the early stages of PGC development. Although several mutations are known to severely reduce the number of PGCs reaching and populating the genital ridges, the molecular identity of only two of these genes is known: the c-kit receptor protein tyrosine kinase and the c-kit ligand (the steel factor). Herein, we report that mutant mice lacking TIAR, an RNA recognition motif/ribonucleoprotein-type RNA-binding protein highly expressed in PGCs, fail to develop spermatogonia or oogonia. This developmental defect is a consequence of reduced survival of PGCs that migrate to the genital ridge around embryonic day 11.5 (E11.5). The numbers of PGCs populating the genital ridge in TIAR-deficient embryos are severely reduced compared to wild-type embryos by E11.5 and in the mutants PGCs are completely absent at E13.5. Furthermore, TIAR-deficient embryonic stem cells do not proliferate in the absence of exogenous leukemia inhibitory factor in an in vitro methylcellulose culture assay, supporting a role for TIAR in regulating cell proliferation. Because the development of PGCs relies on the action of several growth factors, these results are consistent with a role for TIAR in the expression of a survival factor or survival factor receptor that is essential for PGC development. TIAR-deficient mice thus provide a model system to study molecular mechanisms of PGC development and possibly the basis for some forms of idiopathic infertility.     

The carboxyl-terminal region of protein C is essential for its secretion

We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.     

The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.     

Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA

The requirement of PTB, polypyrimidine tract binding protein, for internal initiation of translation has been tested using an RNA affinity column to deplete rabbit reticulocyte lysates of PTB. The affinity column was prepared by coupling CNBr-activated Sepharose with the segment of the 5'-untranslated region of encephalomyocarditis virus (EMCV) RNA previously shown to bind PTB. Lysates passed through this column were devoid of PTB, and were incapable of internal initiation of translation dependent on the EMCV 5'-untranslated region, while retaining the capacity for translation dependent on ribosome scanning. Full activity for internal initiation was restored by the addition of recombinant PTB at the physiologically relevant concentration of about 5 micrograms/mL. When various PTB deletion mutants were tested, it was found that this activity required virtually the full-length protein. Thus, PTB is an essential protein for internal initiation promoted by the EMCV 5'-untranslated region. However, the PTB-depleted lysate retained the capacity for internal initiation promoted by the 5'-untranslated regions of another cardiovirus, Theiler's murine encephalomyelitis virus, and of the unrelated hepatitis C virus, and in neither case did addition of recombinant PTB stimulate internal initiation. Therefore, PTB is not a universal internal initiation factor that is indispensable in every case of internal ribosome entry.     

The motif V of plum pox potyvirus CI RNA helicase is involved in NTP hydrolysis and is essential for virus RNA replication

The plum pox potyvirus (PPV) protein CI is an RNA helicase whose function in the viral life cycle is still unknown. The CI protein contains seven conserved sequence motifs typical of RNA helicases of the superfamily SF2. We have introduced several individual point mutations into the region coding for motif V of the PPV CI protein and expressed these proteins in Escherichia coli as maltose binding protein fusions. Mutations that abolished RNA helicase activity also disturbed NTP hydrolysis. No mutations affected the RNA binding capacity of the CI protein. These mutations were also introduced in the PPV genome making use of a full-length cDNA clone. Mutant viruses carrying CI proteins with reduced RNA helicase activity replicated very poorly in protoplasts and were unable to infect whole plants without rapid pseudoreversion to wild-type. These results indicate that motif V is involved in the NTP hydrolysis step required for potyvirus RNA helicase activity, and that this activity plays an essential role in virus RNA replication inside the infected cell.     

Specific encapsidation of nodavirus RNAs is mediated through the C terminus of capsid precursor protein alpha

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.     

GTP hydrolysis is essential for protein import into the mitochondrial matrix

Protein import into the innermost compartment of mitochondria (the matrix) requires a membrane potential (delta psi) across the inner membrane, as well as ATP-dependent interactions with chaperones in the matrix and cytosol. The role of nucleoside triphosphates other than ATP during import into the matrix, however, remains to be determined. Import of urea-denatured precursors does not require cytosolic chaperones. We have therefore used a purified and urea-denatured preprotein in our import assays to bypass the requirement of external ATP. Using this modified system, we demonstrate that GTP stimulates protein import into the matrix; the stimulatory effect is directly mediated by GTP hydrolysis and does not result from conversion of GTP to ATP. Both external GTP and matrix ATP are necessary; neither one can substitute for the other if efficient import is to be achieved. These results suggest a "push-pull" mechanism of import, which may be common to other post-translational translocation pathways.     

The Myb leucine zipper is essential for leukemogenicity of the v-Myb protein

The AMV v-Myb oncoprotein causes oncogenic transformation of myelomonocytic cells in vivo and in vitro. Its transforming capacity is strictly dependent upon the N-terminal DNA binding domain, the central transactivation region, and on the C-terminal domain containing a putative leucine zipper motif. Here we show that the v-MybL3,4A mutant, in which Leu325 and Leu332 of the leucine zipper have been replaced by alanines, failed to induce leukemia in virus infected chicken. This demonstrates that the leucine zipper domain is indispensable for v-myb induced leukemogenesis in vivo. v-MybL3,4A was, however, still able to transform myelomonocytic cells from chicken bone marrow in vitro. Yet, while v-mybL3,4A transformed cells were impaired in growth at 37 degrees C, they failed to grow at 42 degrees C, the physiological body temperature of avian species. This might explain the loss of v-MybL3,4A leukemogenic potential in vivo. We also demonstrate that the v-Myb leucine zipper domain interacts in vitro with two host cell proteins, p26 and p28. This interaction is compromised in v-MybL3,4A indicating that binding of v-Myb to p26 and p28 might be important for the leukemogenic potential of v-Myb.     

The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli

The rpoS-encoded sigma(S) subunit of RNA polymerase in Escherichia coli is a global regulatory factor involved in several stress responses. Mainly because of increased rpoS translation and stabilization of sigma(S), which in nonstressed cells is a highly unstable protein, the cellular sigma(S) content increases during entry into stationary phase and in response to hyperosmolarity. Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Qbeta RNA, as an essential factor for rpoS translation. An hfq null mutant exhibits strongly reduced sigma(S) levels under all conditions tested and is deficient for growth phase-related and osmotic induction of sigma(S). Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation. We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I. In addition, we show that expression and heat induction of the heat shock sigma factor sigma(32) (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure. HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E. coli.     

NUP82 is an essential yeast nucleoporin required for poly(A)+ RNA export

We have isolated and characterized the gene encoding a novel essential nucleoporin of 82 kD, termed NUP82. Indirect immunofluorescence of cells containing an epitope tagged copy of the NUP82 localized it to the nuclear pore complex (NPC). Primary structure analysis indicates that the COOH-terminal 195 amino acids contain a putative coiled-coil domain. Deletion of the COOH-terminal 87 amino acids of this domain causes slower cell growth; deletion of the COOH-terminal 108 amino acids results in slower growth at 30 degrees C and lethality at 37 degrees C. Cells in which the last 108 amino acids of NUP82 have been deleted, when shifted to 37 degrees C, do not display any gross morphological defects in their nuclear pore complexes or nuclear envelopes. They do, however, accumulate poly(A)+ RNA in their nuclei at 37 degrees C. We propose that NUP82 acts as a linker to tether nucleoporins directly involved in nuclear transport to pore scaffolding via its coiled-coil domain.