Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes

In eukaryotic cells, it is generally accepted that protein synthesis is compartmentalized; soluble proteins are synthesized on free ribosomes, whereas secretory and membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes. The partitioning of mRNAs that accompanies such compartmentalization arises early in protein synthesis, when ribosomes engaged in the translation of mRNAs encoding signal-sequence-bearing proteins are targeted to the ER. In this report, we use multiple cell fractionation protocols, in combination with cDNA microarray, nuclease protection, and Northern blot analyses, to assess the distribution of mRNAs between free and ER-bound ribosomes. We find a broad representation of mRNAs encoding soluble proteins in the ER fraction, with a subset of such mRNAs displaying substantial ER partitioning. In addition, we present evidence that membrane-bound ribosomes engage in the translation of mRNAs encoding soluble proteins. Single-cell in situ hybridization analysis of the subcellular distribution of mRNAs encoding ER-localized and soluble proteins identify two overall patterns of mRNA distribution in the cell-endoplasmic reticular and cytosolic. However, both partitioning patterns include a distinct perinuclear component. These results identify previously unappreciated roles for membrane-bound ribosomes in the subcellular compartmentalization of protein synthesis and indicate possible functions for the perinuclear membrane domain in mRNA sorting in the cell.     

Possible utilization of -1 Ribosomal frame shifting in the expression of a human SEMA6C isoform

We have used bioinformatics approaches to identify a potential case of -1 ribosomal frame shifting in the mRNAs of the three variants of human SEMA6C protein. The mRNAs contain a heptanucleotide slippery sequence followed by a compact H-type pseudoknot. Unlike -1 frameshifting signals in viral or viral-like mRNAs, the slippery sequence and downstream pseudoknot in SEMA6C mRNAs locate 423 nucleotides (encoding 141 amino acids) upstream of the stop codon. The potential -1 frameshifting event would produce a polypeptide of 238 residues encoded by the -1 reading frames. Sequence similarity searches using BLAST indicate that ~90% of the 238 residues match actual protein sequences annotated as SEMA6C proteins in the database. We propose that the mRNAs of human SEMA6C utilize a pseudoknot dependent -1 ribosomal frameshifting mechanism to express novel SEMA6C isoforms.     

Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs.

The essential products of the yeast mitochondrial translation system are seven hydrophobic membrane proteins and Var1p, a hydrophilic protein in the small ribosomal subunit. Translation of the membrane proteins depends on nuclearly encoded, mRNA-specific translational activators that recognize the 5'-untranslated leaders of their target mRNAs. These translational activators are themselves membrane associated and could therefore tether translation to the inner membrane. In this study, we tested whether chimeric mRNAs with the untranslated sequences normally present on the mRNA encoding soluble Var1p, can direct functional expression of coding sequences specifying the integral membrane proteins Cox2p and Cox3p. DNA sequences specifying these chimeric mRNAs were inserted into mtDNA at the VAR1 locus and expressed in strains containing a nuclearly localized plasmid that supplies a functional form of Var1p, imported from the cytoplasm. Although cells expressing these chimeric mRNAs actively synthesized both membrane proteins, they were severely deficient in cytochrome c oxidase activity and in the accumulation of Cox2p and Cox3p, respectively. These data strongly support the physiological importance of interactions between membrane-bound mRNA-specific translational activators and the native 5'-untranslated leaders of the COX2 and COX3 mRNAs for localizing productive synthesis of Cox2p and Cox3p to the inner membrane.     

Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.     

Alternative splice variants encoding unstable protein domains exist in the human brain

Alternative splicing has been recognized as a major mechanism by which protein diversity is increased without significantly increasing genome size in animals and has crucial medical implications, as many alternative splice variants are known to cause diseases. Despite the importance of knowing what structural changes alternative splicing introduces to the encoded proteins for the consideration of its significance, the problem has not been adequately explored. Therefore, we systematically examined the structures of the proteins encoded by the alternative splice variants in the HUGE protein database derived from long (>4 kb) human brain cDNAs. Limiting our analyses to reliable alternative splice junctions, we found alternative splice junctions to have a slight tendency to avoid the interior of SCOP domains and a strong statistically significant tendency to coincide with SCOP domain boundaries. These findings reflect the occurrence of some alternative splicing events that utilize protein structural units as a cassette. However, 50 cases were identified in which SCOP domains are disrupted in the middle by alternative splicing. In six of the cases, insertions are introduced at the molecular surface, presumably affecting protein functions, while in 11 of the cases alternatively spliced variants were found to encode pairs of stable and unstable proteins. The mRNAs encoding such unstable proteins are much less abundant than those encoding stable proteins and tend not to have corresponding mRNAs in non-primate species. We propose that most unstable proteins encoded by alternative splice variants lack normal functions and are an evolutionary dead-end.     

Codon usage in Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma)

By sequence analysis of 96 randomly selected clones in a cDNA library of Xanthophyllomyces dendrorhous, ten novel, full-length clones encoding cytoplasmic ribosomal proteins (rp) were found. The deduced amino acid sequences showed significant homology to their counterparts from eukaryotic origin including mammals, fungi and plants. Some ribosmal protein encoding cDNAs appeared several times, but by Southern blot analysis it was shown they are encoded by a single copy gene. The nucleotide sequences of ten full length cDNAs were used to investigate the codon usage in X. dendrorhous.     

Cytokinin stimulates polyribosome loading of nuclear-encoded mRNAs for the plastid ATP synthase in etioplasts of Lupinus luteus: the complex accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space

Three of the nine subunits of the plastid ATP synthase, including the subunit of the CF(1) moiety (gene AtpC), are encoded in the nucleus. Application of cytokinin to etiolated lupine seedlings induces polyribosome association of their mRNAs. This appears to be specific as no such regulation was observed for messages for three ribosomal proteins. Cytokinin-mediated polyribosome loading was also observed for the spinach AtpC message in etiolated transgenic tobacco seedlings. Analysis of various spinach AtpC mRNA derivatives uncovered that the 5' untranslated region (5' UTR) of this message is sufficient to direct polyribosome loading, and that sequences at the 3' end of the AtpC 5' UTR, including an UC-rich motif, are crucial for this regulation. The increase in polyribosome loading of the AtpC message correlated with an increased synthesis of the polypeptide. The subunit, together with the ATP synthase complex, accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space of the etioplast. These results suggest that cytokinin promotes accumulation of the ATP synthase in the inner-envelope membrane of lupine etioplasts by stimulating the translation efficiency of their nuclear-encoded messages.     

An Integrated Sequence-Structure Database incorporating matching mRNA sequence, amino acid sequence and protein three-dimensional structure data.

We have constructed a non-homologous database, termed the Integrated Sequence-Structure Database (ISSD) which comprises the coding sequences of genes, amino acid sequences of the corresponding proteins, their secondary structure and straight phi,psi angles assignments, and polypeptide backbone coordinates. Each protein entry in the database holds the alignment of nucleotide sequence, amino acid sequence and the PDB three-dimensional structure data. The nucleotide and amino acid sequences for each entry are selected on the basis of exact matches of the source organism and cell environment. The current version 1.0 of ISSD is available on the WWW at http://www.protein.bio.msu.su/issd/ and includes 107 non-homologous mammalian proteins, of which 80 are human proteins. The database has been used by us for the analysis of synonymous codon usage patterns in mRNA sequences showing their correlation with the three-dimensional structure features in the encoded proteins. Possible ISSD applications include optimisation of protein expression, improvement of the protein structure prediction accuracy, and analysis of evolutionary aspects of the nucleotide sequence-protein structure relationship.     

Localization of nuclear-encoded mRNAs to mitochondria outer surface

The diverse functions of mitochondria depend on hundreds of different proteins. The vast majority of these proteins is encoded in the nucleus, translated in the cytosol, and must be imported into the organelle. Import was shown to occur after complete synthesis of the protein, with the assistance of cytosolic chaperones that maintain it in an unfolded state and target it to the mitochondrial translocase of the outer membrane (TOM complex). Recent studies, however, identified many mRNAs encoding mitochondrial proteins near the outer membrane of mitochondria. Translation studies suggest that many of these mRNAs are translated locally, presumably allowing cotranslational import into mitochondria. Herein we review these data and discuss its relevance for local protein synthesis. We also suggest alternative roles for mRNA localization to mitochondria. Finally, we suggest future research directions, including revealing the significance of localization to mitochondria physiology and the molecular players that regulate it.     

Localization of mRNAs encoding human mitochondrial oxidative phosphorylation proteins

The mitochondrial oxidative phosphorylation (OXPHOS) proteins are encoded by both nuclear and mitochondrial DNA. The nuclear-encoded OXPHOS mRNAs have specific subcellular localizations, but little is known about which localize near mitochondria. Here, we compared mRNAs in mitochondria-bound polysome fractions with those in cytosolic, free polysome fractions. mRNAs encoding hydrophobic OXPHOS proteins, which insert into the inner membrane, were localized near mitochondria. Conversely, OXPHOS gene which mRNAs were predominantly localized in cytosol had less than one transmembrane domain. The RNA-binding protein Y-box binding protein-1 is localized at the mitochondrial outer membrane and bound to the OXPHOS mRNAs. Our findings offer new insight into mitochondrial co-translational import in human cells.     

Correlation Between Shine–Dalgarno Sequence Conservation and Codon Usage of Bacterial Genes

In this study, we analyzed the correlation between codon usage bias and Shine–Dalgarno (SD) sequence conservation, using complete genome sequences of nine prokaryotes. For codon usage bias, we adopted the codon adaptation index (CAI), which is based on the codon usage preference of genes encoding ribosomal proteins, elongation factors, heat shock proteins, outer membrane proteins, and RNA polymerase subunit proteins. To compute SD sequence conservation, we used SD motif sequences predicted by Tompa and systematically aligned them with 5′UTR sequences. We found that there exists a clear correlation between the CAI values and SD sequence conservation in the genomes of Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii, and no relationship is found in M. genitalium, M. pneumoniae, and Synechocystis. That is, genes with higher CAI values tend to have more conserved SD sequences than do genes with lower CAI values in these organisms. Some organisms, such as M. thermoautotrophicum, do not clearly show the correlation. The biological significance of these results is discussed in the context of the translation initiation process and translation efficiency. Received: 22 June 2000 / Accepted: 18 October 2000