A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs

In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using (75)Se-labeled Sec-tRNA(Sec), we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co-translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.     

A nuclear-encoded function essential for translation of the chloroplast psaB mRNA in chlamydomonas.

We report the analysis of a photosystem I-deficient mutant of Chlamydomonas reinhardtii, F15, that contains a mutation at the TAB1 (for translation of psaB mRNA) nuclear locus. Pulse labeling of chloroplast proteins revealed that the synthesis of the two photosystem I reaction center polypeptides PSAA and PSAB was undetectable in this mutant. The mRNA levels of these proteins were only moderately reduced, suggesting that the primary defect occurs at a step during or after translation. We constructed chimeric genes consisting of the psaA and psaB 5' untranslated region (5' UTR) fused to the aminoglycoside adenyltransferase (aadA) coding sequence, which confers spectinomycin resistance. Insertion of these genes into the chloroplast genome through biolistic transformation and analysis of their expression in the TAB1 mutant nuclear background revealed that the psaB (but not the psaA) 5' UTR is the target of the wild-type TAB1 function. This suggests that TAB1 is required for the initiation of psaB mRNA translation. The dependence of PSAA synthesis or accumulation on PSAB synthesis is strongly suggested by the identification of a suppressor mutation within the psaB 5' UTR. The suppressor specifically restores the synthesis of both proteins in the presence of the tab1-F15 mutation. The location of the suppressor mutation within a putative base-paired region near the psaB initiation codon suggests a role for TAB1 in the activation of translation of the psaB mRNA.     

CLA1, a novel gene required for chloroplast development, is highly conserved in evolution

An albino mutant designated cla1-1 (for ‘c loroplastos a lterado’, or ‘altered chloroplasts’) has been isolated from a T-DNA-generated library of Arabidopsis thaliana. In cla11 plants, chloroplast development is arrested at an early stage. cla1-1 plants behave like wild-type in their capacity to etiolate and produce anthocyanins indicating that the light signal transduction pathway seems to be unaffected. Genetic and molecular analyses show that the disruption of a single gene, CLA1, by the T-DNA insertion is responsible for the mutant phenotype. RNA expression patterns indicate that CLA1 is positively regulated by light and that it has different effects on the steady-state RNA levels of some nuclear- and chloroplast-encoded photosynthetic genes. Although the specific function of the CLA1 gene is still unknown, it encodes a novel protein conserved in evolution between photosynthetic bacteria and plants which is essential for chloroplast development in Arabidopsis.     

The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro

Metazoan replication-dependent histone mRNAs end in a conserved stem-loop rather than in the poly(A) tail found on all other mRNAs. The 3' end of histone mRNA binds a single class of proteins, the stem-loop binding proteins (SLBP). In Xenopus, there are two SLBPs: xSLBP1, the homologue of the mammalian SLBP, which is required for processing of histone pre-mRNA, and xSLBP2, which is expressed only during oogenesis and is bound to the stored histone mRNA in Xenopus oocytes. The stem-loop is required for efficient translation of histone mRNAs and substitutes for the poly(A) tail, which is required for efficient translation of other eucaryotic mRNAs. When a rabbit reticulocyte lysate is programmed with uncapped luciferase mRNA ending in the histone stem-loop, there is a three- to sixfold increase in translation in the presence of xSLBP1 while xSLBP2 has no effect on translation. Neither SLBP affected the translation of a luciferase mRNA ending in a mutant stem-loop that does not bind SLBP. Capped luciferase mRNAs ending in the stem-loop were injected into Xenopus oocytes after overexpression of either xSLBP1 or xSLBP2. Overexpression of xSLBP1 in the oocytes stimulated translation, while overexpression of xSLBP2 reduced translation of the luciferase mRNA ending in the histone stem-loop. A small region in the N-terminal portion of xSLBP1 is required to stimulate translation both in vivo and in vitro. An MS2-human SLBP1 fusion protein can activate translation of a reporter mRNA ending in an MS2 binding site, indicating that xSLBP1 only needs to be recruited to the 3' end of the mRNA but does not need to be directly bound to the histone stem-loop to activate translation.     

SLIP1, a factor required for activation of histone mRNA translation by the stem-loop binding protein

Replication-dependent histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated, ending instead in a conserved stem-loop. The 3′ end of histone mRNA is required for histone mRNA translation, as is the stem-loop binding protein (SLBP), which binds the 3′ end of histone mRNA. We have identified five conserved residues in a 15-amino-acid region in the amino-terminal portion of SLBP, each of which is required for translation. Using a yeast two-hybrid screen, we identified a novel protein, SLBP-interacting protein 1 (SLIP1), that specifically interacts with this region. Mutations in any of the residues required for translation reduces SLIP1 binding to SLBP. The expression of SLIP1 in Xenopus oocytes together with human SLBP stimulates translation of a reporter mRNA ending in the stem-loop but not a reporter with a poly(A) tail. The expression of SLIP1 in HeLa cells also stimulates the expression of a green fluorescent protein reporter mRNA ending in a stem-loop. RNA interference-mediated downregulation of endogenous SLIP1 reduces the rate of translation of endogenous histone mRNA and also reduces cell viability. SLIP1 may function by bridging the 3′ end of the histone mRNA with the 5′ end of the mRNA, similar to the mechanism of translation of polyadenylated mRNAs.     

The conserved 5 S rRNA complement to tRNA is not required for translation of natural mRNA

We have tested a putative base-paired interaction between the conserved GT psi C sequence of tRNA and the conserved GAAC47 sequence of 5 S ribosomal RNA by in vitro protein synthesis using ribosomes containing deletions in this region of 5 S rRNA. Ribosomes reconstituted with 5 S rRNA possessing a single break between residues 41 and 42, deletion of residues 42-46, or deletion of residues 42-52 were tested for their ability to translate phage MS2 RNA. Initiator tRNA binding, aminoacyl-tRNA binding, ppGpp synthesis, and miscoding were also tested. All of the measured functions could be carried out by ribosomes carrying the deleted 5 S rRNAs. The sizes and relative amounts of the polypeptides synthesized by MS2 RNA-programmed ribosomes were identical whether or not the 5 S RNA contained deletions. Aminoacyl-tRNA binding and miscoding were essentially unaffected. Significant reduction in ApUpG (but not poly(A,U,G) or MS2 RNA)-directed fMet-tRNA binding and ppGpp synthesis were observed, particularly in the case of the larger (residues 42-52) deletion. We conclude that if tRNA and 5 S rRNA interact in this fashion, it is not an obligatory step in protein synthesis.     

Identification and characterization of a novel RNA binding protein that associates with the 5'-untranslated region of the chloroplast psbA mRNA

Binding of proteins to chloroplast-encoded mRNAs has been shown to be an essential part of chloroplast gene expression. Four nuclear-encoded proteins (38, 47, 55, and 60 kDa) have been identified that bind to the 5'-untranslated region of the Chlamydomonas reinhardtii psbA mRNA with high affinity and specificity. We have cloned a cDNA that represents the 38 kDa protein (RB38) and show that it encodes a novel RNA binding protein that is primarily localized within the chloroplast stroma. RB38 contains four 70 amino acid repeats with a high percentage of basic amino acids, as well as an amino-terminal extension predicted to act as a chloroplast import sequence. We demonstrate that the 38 kDa precursor protein is imported into isolated chloroplasts and interacts with high specificity to uridine-rich regions within the 5'-untranslated region of the psbA mRNA. While database searches have identified hypothetical proteins from several other eukaryotic species with high sequence similarity to the deduced amino acid sequence of RB38, no proteins with homology to RB38 have been biochemically characterized. Bioinformatic analysis of the RB38 sequence, together with structure analysis using circular dichroism and protein modeling, suggests that the 70 amino acid repeats within RB38 are similar in fold to previously identified RNA binding motifs, despite limited sequence homology.     

Both RNA editing and RNA cleavage are required for translation of tobacco chloroplast ndhD mRNA: a possible regulatory mechanism for the expression of a chloroplast operon consisting of functionally unrelated genes.

Tobacco chloroplast genes encoding a photosystem I component (psaC) and a NADH dehydrogenase subunit (ndhD) are transcribed as a dicistronic pre-mRNA which is then cleaved into short mRNAs. An RNA protection assay revealed that the cleavage occurs at multiple sites in the intercistronic region. There are two possible initiation codons in the tobacco ndhD mRNA: the upstream AUG and the AUG created by RNA editing from the in-frame ACG located 25 nt downstream. Using the chloroplast in vitro translation system, we found that translation begins only from the edited AUG. The extent of ACG to AUG editing is partial and depends on developmental and environmental conditions. In addition, the in vitro assay showed that the psaC/ndhD dicistronic mRNA is not functional and that the intercistronic cleavage is a prerequisite for both ndhD and psaC translation. Using a series of mutant mRNAs, we showed that an intramolecular interaction between an 8 nt sequence in the psaC coding region and its complementary 8 nt sequence in the 5' ndhD UTR is the negative element for translation of the dicistronic mRNA. A possible mechanism in which the differential expression of the chloroplast operon consists of functionally unrelated genes is discussed.     

Characterization of Tbc2, a nucleus-encoded factor specifically required for translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii

Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are involved in the translation of the chloroplast psbC mRNA of the eukaryotic green alga Chlamydomonas reinhardtii. In this study we report the isolation and phenotypic characterization of two new tbc2 mutant alleles and their use for cloning and characterizing the Tbc2 gene by genomic complementation. TBC2 encodes a protein of 1,115 residues containing nine copies of a novel degenerate 38-40 amino acid repeat with a quasiconserved PPPEW motif near its COOH-terminal end. The middle part of the Tbc2 protein displays partial amino acid sequence identity with Crp1, a protein from Zea mays that is implicated in the processing and translation of the chloroplast petA and petD RNAs. The Tbc2 protein is enriched in chloroplast stromal subfractions and is associated with a 400-kD protein complex that appears to play a role in the translation of specifically the psbC mRNA.     

Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES

The cellular protein, poly(rC) binding protein 2 (PCBP2), is known to function in picornavirus cap-independent translation. We have further examined the RNA binding properties and protein-protein interactions of PCBP2 necessary for translation. We have studied its putative multimerization properties utilizing the yeast two-hybrid assay and in vitro biochemical methods, including glutathione S-transferase (GST) pull-down assays and gel filtration. Through genetic analysis, the multimerization domain has been localized to the second K-homologous (KH) RNA binding domain of the protein between amino acids 125 and 158. To examine the function of multimerization in poliovirus translation, we utilized the truncated protein, DeltaKH1-PCBP2, which is capable of multimer formation, but does not bind poliovirus stem-loop IV RNA (an interaction required for translation). Utilizing RNA binding and in vitro translation assays, this protein was shown to act as a dominant negative, suggesting that PCBP2 multimerization functions in poliovirus translation and RNA binding. Additionally, PCBP2 containing a deletion in the multimerization domain (DeltaKH2-PCBP2) was not able to bind poliovirus stem-loop IV RNA and could not rescue translation in extracts that were depleted of endogenous PCBP2. Results from these experiments suggest that the multimerization of PCBP2 is required for efficient RNA binding and cap-independent translation of poliovirus RNA. By examining the functional interactions of the cellular protein PCBP2, we have discovered a novel determinant in the mechanism of picornavirus cap-independent translation.     

CSP41, a sequence-specific chloroplast mRNA binding protein, is an endoribonuclease.

Correct 3' processing of chloroplast precursor mRNAs (pre-mRNAs) requires a stem-loop structure within the 3' untranslated region. In spinach, a stable 3' stem-loop-protein complex has been shown to form in vitro between petD pre-mRNA, encoding subunit IV of the cytochrome b6/f complex, and chloroplast proteins. This complex contains three chloroplast stem-loop binding proteins (CSPs), namely, CSP29, CSP41, and CSP55. Here, we report the purification of CSP41 and cloning of the csp41 gene and show that CSP41 is encoded by a single nuclear gene. Characterization of bacterially expressed CSP41 demonstrates that this protein binds specifically to the 3' stem-loop structure and a downstream AU-rich element of petD pre-mRNA and that its binding affinity is enhanced by associating with CSP55. Our data also show that CSP41 has substantial nonspecific endoribonuclease activity. These data suggest that CSP41 could be involved in 3' processing of petD pre-mRNA and/or in RNA degradation. The fact that different reaction conditions favor RNA binding over ribonuclease activities suggests a possible mode of in vivo regulation.     

She2p is a novel RNA binding protein with a basic helical hairpin motif

Selective transport of mRNAs in ribonucleoprotein particles (mRNP) ensures asymmetric distribution of information within and among eukaryotic cells. Actin-dependent transport of ASH1 mRNA in yeast represents one of the best-characterized examples of mRNP translocation. Formation of the ASH1 mRNP requires recognition of zip code elements by the RNA binding protein She2p. We determined the X-ray structure of She2p at 1.95 A resolution. She2p is a member of a previously unknown class of nucleic acid binding proteins, composed of a single globular domain with a five alpha helix bundle that forms a symmetric homodimer. After demonstrating potent, dimer-dependent RNA binding in vitro, we mapped the RNA binding surface of She2p to a basic helical hairpin in vitro and in vivo and present a mechanism for mRNA-dependent initiation of ASH1 mRNP complex assembly.     

Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro

Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. Here we describe cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, we demonstrate that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor.