Determination of the secondary structure of and cellular protein binding to the 3'-untranslated region of the hepatitis C virus RNA genome.

Hepatitis C virus (HCV) contains a positive-stranded RNA genome of approximately 9.5 kb. Despite the overall sequence diversity among individual HCV isolates, the 3'-end 98 nucleotides (nt) of the HCV RNA, which constitute part of the 3'-untranslated region (3'-UTR), are highly conserved. This conserved region may contain the cis-acting signals for RNA replication involving possibly both viral and cellular proteins. We carried out RNase digestion studies, which revealed that this 98-nt region contains three stem-loops but may also assume alternative structures. We further performed UV cross-linking experiments to detect cellular proteins that bound to this region. A 58-kDa cellular protein (p58) was detected. Its binding site was mapped to the stem-loops 2 and 3, which are the most conserved region of the 3'-UTR. Site-directed mutagenesis studies revealed that both stem structures and specific nucleotide sequence within the two loops are important for p58 binding. Mutations that disrupted stem structures abolished protein binding, while the compensatory mutations restored its binding. This region also contains partial sequence similarity to the reported consensus binding sequence for polypyrimidine tract-binding protein (PTB) (a 57-kDa protein). The UV-cross-linked protein could be immunoprecipitated with the anti-PTB antibody, and the recombinant PTB bound to the HCV 3'-UTR with the same binding specificity as p58, establishing that this protein is PTB. However, the reported PTB-binding sequence was not sufficient, but rather the entire stem-loops 2 and 3 were required, for PTB binding; thus, its binding specificity is significantly different from the reported PTB-binding sequence requirement. This protein was detected in both the nuclei and cytoplasm of most mammalian cell lines tested and human primary hepatocytes. PTB may participate in the regulation of HCV RNA synthesis or translation.     

5' and 3' untranslated regions of pestivirus genome: primary and secondary structure analyses.

Within the conserved 5' untranslated region (UTR) of the pestivirus genome three highly variable regions were identified. Preceding the polyprotein start codon, multiple cryptic AUG codons and several small open reading frames are characteristic for all the five pestiviruses. Inspection of the context of AUGs revealed that the polyprotein initiation AUG of pestivirus has a weak context for efficient translation initiation. The most favorable context was found in two of the cryptic AUGs. Two oligopyrimidine-rich tracts upstream to the conserved either cryptic or authentic AUG in the 5'-UTR of pestivirus were identified and 83.3% of their nucleotide sequences are complementary to the consensus sequence at the 3' terminus of eucaryotic 18S rRNA. A secondary structure model for the 5'-UTR of pestivirus was predicted. Nucleotide sequence comparison among five pestiviruses led to the identification of a variable region and a conserved region in the 3'-UTR. A deletion of 41 nucleotides was found within the variable region in Osloss. A secondary structure model for the 3'-UTR was also predicted. The structural similarity of the 5'-UTR between pestiviruses and picornaviruses and hepatitis C viruses was demonstrated and the possible implications of features of the 5' and 3'-UTR of pestiviruses are discussed.     

The proximal region of the 3'-untranslated region of cyclooxygenase-2 is recognized by a multimeric protein complex containing HuR,TIA-1, TIAR,and the heterogeneous nuclear ribonucleoprotein U

Cyclooxygenase-2 (COX-2) is an early response gene induced in renal mesangial cells by interleukin-1beta (IL-1beta). The 3'-untranslated region (3'-UTR) of COX-2 mRNA plays an important role in IL-1beta induction by regulating message stability and translational efficiency. The first 60 nucleotides of the 3'-UTR of COX-2 are highly conserved and contain multiple copies of the regulatory sequence AUUUA. Introduction of the 60-nucleotide sequence into the 3'-UTR of a heterologous reporter gene resulted in a 70% decrease in reporter gene expression. Electrophoretic mobility shift assays (EMSAs) demonstrated that mesangial cell nuclear fractions contain a multimeric protein complex that bound this region of COX-2 mRNA in a sequence-specific manner. We identified four members of the protein-RNA complex as HuR, TIA-1, TIAR, and the heterogeneous nuclear ribonucleoprotein U (hnRNP U). Treatment of mesangial cells with IL-1beta caused an increase in cytosolic HuR, which was accompanied by an increase in COX-2 mRNA that co-immunoprecipitated with cytosolic HuR. Therefore, we propose that HuR binds to the proximal region of the 3'-UTR of COX-2 following stimulation by IL-1beta and increases the expression of COX-2 mRNA by facilitating its transport out of the nucleus.     

Specificity of the STAR/GSG domain protein Qk1: implications for the regulation of myelination

Inadequate formation and maintenance of myelin is the basis for several neurodegenerative disorders, including leukodystrophy and multiple sclerosis. In mice, oligodendrocyte differentiation and subsequent formation of myelin requires the Quaking gene. Mutation of this gene leads to embryonic lethality or to a trembling phenotype characteristic of dysmyelination. Quaking encodes Qk1, a member of the highly conserved STAR/GSG family of RNA-binding proteins that function as master developmental regulators in higher eukaryotes. Qk1 has been implicated in the regulation of alternative splicing, stability, and translation control of mRNAs that code for myelin structural components in glial cells. We have used quantitative gel mobility shift and fluorescence polarization assays to define the nucleotide sequence specificity of the Qk1 STAR/GSG domain, and to probe the interaction between Qk1 and the 3'-untranslated region (UTR) of myelin basic protein (MBP) mRNA. The results show that Qk1 recognizes a hexanucleotide consensus element that is similar although not identical to the specificity determinant recognized by the Caenorhabditis elegans STAR/GSG protein GLD-1. Several consensus sites are present in the 3'-UTR of MBP mRNA. The highest affinity site is located within the RNA localization region, suggesting a possible role for Qk1 in restricting MBP mRNA to the myelin compartment.     

Nucleotide sequence analysis of cDNA encoding the coat protein of cucumber mosaic virus: genome organization and molecular features of the protein

The cDNA sequence coding for the coat protein of cucumber mosaic virus (Japanese Y strain) was cloned, and its nucleotide sequence was determined. The sequence contains an open reading frame that encodes the coat protein composed of 218 amino acids. The nucleotide and deduced amino acid sequences of the coat protein of this strain were compared with those of the Q strain; the homologies of the sequences were 78% and 81%, respectively. Further study of the sequences gave an insight into the genome organization and the molecular features of the coat protein. The coding region can be divided into three characteristic regions. The N-terminal region has conserved features in the positively charged structure, the hydropathy pattern and the predicted secondary structure, although the amino acid sequence is varied mainly due to frameshift mutations. It is noteworthy that the positions of arginine residues in this region are highly conserved. Both the nucleotide and amino acid sequences of the central region are well conserved. The amino acid sequence of the C-terminal region is not conserved, because of frameshift mutations, however, the total number of amino acids is conserved. The nucleotide sequence of the 3'-noncoding region is divergent, but it could form a tRNA-like structure similar to those reported for other viruses. Detailed investigation suggests that the Y and Q strains are evolutionarily distant.     

Molecular cloning of the murine branched chain α-ketoacid dehydrogenase E2 subunit: presence of 3′ B1 repeat elements

The cDNA sequence encoding the murine E2 subunit (dihydrolipoyl transacylase) of the branched-chain α-ketoacid dehydrogenase (BCKAD) complex was determined. In the region encoding the mature E2 subunit protein, both the nucleotide composition and predicted amino acid sequence are highly conserved between murine, human, and bovine species. In contrast, the 5′ sequence encoding the amino-terminal preprotein sequence and 3′ untranslated region are less well conserved. The 3′-noncoding region contains sequences highly homologous to the rodent B1 repeat elements, which are related to human Alu repeat sequences. This finding is similar to the presence of three Alu repeat sequences in the 3′-noncoding region of human E2 cDNA.     

Identification of a GA-rich sequence as a protein-binding site in the 3'-untranslated region of chicken elastin mRNA with a potential role in the developmental regulation of elastin mRNA stability

Synthesis of aortic elastin peaks in the perinatal period and then is strongly down-regulated with postnatal development and growth. Decreased stability of elastin mRNA contributes to this developmental decrease in chick aortic elastin production. We have previously shown that destabilization of elastin mRNA is correlated with decreased binding of cytosolic protein(s) to a large, GC-rich region of secondary structure in the 3'-untranslated region (3'-UTR) of elastin mRNA. In this study, using gel migration shift assays, deletion constructs, and antisense competition assays, we identify a major protein-binding site in the 3'-UTR of elastin as a GA-rich sequence (UGGGGGGAGGGAGGGAGGGA), which we have designated the G3A motif. This motif is present in the 3'-UTR of elastin from several species. Binding proteins are present in both nuclear and cytoplasmic extracts, and their abundance is associated with tissues producing elastin and correlated with circumstances in which elastin mRNA is stable. These results suggest that the conserved GA-rich sequence of the elastin 3'-UTR is an important element in the regulation of stability of the elastin mRNA.     

猪繁殖与呼吸障碍综合征病毒(PRRSV)3′末端非翻译区中调控序列的研究

以北美株PRRSV感染性克隆pCBC2为平台进行反向遗传操作,将3′UTR中的一级结构进行了系列缺失或插入突变,并改变二级结构中的一个保守的茎环结构,构建全长PRRSV突变体克隆,解析3′UTR突变对病毒感染性的影响,旨在界定调控PRRSV3′UTR的启动子序列及二级结构,即复制过程中的最小调控元件。以空斑和Northern blot来研究拯救后重组病毒的复制、转录和生长特性,发现重组病毒感染动力学与亲本病毒无可见差别。结果表明PRRSV3′UTR的5′端可耐受一定数目的核苷酸的缺失(41nt)与插入(23nt)突变,但进一步9nt缺失造成保守的环结构突变后就使病毒失去了感染性。证明了这是3′UTR中控制PRRSV复制过程的的必需序列及二级结构,为进一步解析PRRSV复制过程的调控元件奠定了基础。     

Molecular cloning of the mouse S-adenosylmethionine decarboxylase cDNA: specific protein binding to the conserved region of the mRNA 5'-untranslated region.

The nucleotide sequence of a cDNA encoding the proenzyme of mouse S-adenosylmethionine decarboxylase (AdoMetDC) including 257 nucleotides of the 5' untranslated region has been determined. Comparison of the nucleotide sequence of the mouse 5' untranslated region with those of other mammals shows it to be highly conserved. The 52 nucleotides upstream from the translation initiation codon are identical in human, rat, bovine and mouse. The polyamines, spermidine and spermine, have been shown to inhibit AdoMetDC mRNA translation. An RNA gel retardation assay demonstrated that a cytoplasmic extract from mouse brain forms an RNA-protein complex with the completely conserved 5' untranslated sequence and that the complex formation is highly dependent on the presence of spermine. Crosslinking by UV irradiation shows that the complex contains a 39-kDa protein interacting with the 5' untranslated sequence. These data demonstrate spermine-dependent specific protein binding to a highly conserved 5' untranslated region of an mRNA translationally regulated by polyamines.     

Conserved and unique sequences in the 3'-untranslated region of rat smooth-muscle alpha-actin mRNA

We have isolated a cDNA clone corresponding to rat smooth-muscle alpha-actin mRNA [Hsu and Frankel, J. Biol. Chem. 262 (1987) 9594-9600]. We present here the sequence of the 3'-untranslated region (3'-UTR) of the cDNA. By comparison with the reported sequence of the chicken gene, this 3'-UTR region contains a conserved 36-bp sequence and a unique 48-bp G + C-rich sequence. An RNA probe containing only the 3'-UTR of the cDNA was synthesized and shown to be specific for smooth-muscle alpha-actin message.     

Molecular cloning and nucleotide sequence of the 30K and the coat protein cistron of TMV (tomato strain) genome.

The cDNA copies of tobacco mosaic virus (TMV)-tomato strain (L) genome were cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2, 161-170. (1982)) and the sequence of 1,614 nucleotides at the 3' end was determined. The sequence encompasses the 30K and the coat protein cistron which are located in residues 685-1, 479 and 203-682 from the 3' end of the genome respectively. The close relationship between the tomato and the common strain was shown on the level of the nucleotide sequence. Highly homologous regions are found in the 3' non-coding region, the assembly origin and the 5' flanking region of the 30K protein cistron. The comparison of the deduced amino acid sequence between the tomato and the common strain shows that the 30K protein is composed of the conserved N-terminal four-fifth and the highly divergent region near the C-terminus.     

Genomic structure of the bovine PrP gene and complete nucleotide sequence of bovine PrP cDNA

The present authors previously reported the nucleotide sequence of the 5' half of a cDNA encoding bovine prion protein (PrP) and the genomic structure of the bovine PrP gene encoding the 5'-untranslated region. Here they report the extent of intron 2 of the bovine PrP gene and the nucleotide sequence of the 3' half of bovine PrP cDNA that had not been determined before. This newly sequenced 3' half of the bovine PrP cDNA consisted of 2149 bp. The entire 3'-untranslated region (3'-UTR) was found to be encoded by a single exon, exon 3. One nucleotide polymorphism was found in the 3'-UTR. The length of intron 2 was estimated to be about 14 kbp. The structure of bovine PrP gene can be defined by combining the present results and previous reports on the bovine PrP gene.     

Isolation and characterization of the rat proenkephalin gene

The rat proenkephalin gene has been isolated by molecular cloning and characterized by DNA-sequence analysis. The gene exhibits a structural organization similar to that of the human gene. The nucleotide sequence encoding the biologically active opioid peptides which are generated from the proenkephalin precursor as well as the 3' untranslated region of the mRNA are found on a large exon at the 3' end of the gene (Exon III). The nucleotide sequence encoding the N terminus of the mature protein and its signal peptide are located on Exon II while Exon I encodes the 5' untranslated region of the mRNA. The nucleotide sequence of these exons and their flanking regions has been determined and compared to the human proenkephalin gene. Analysis of the nucleotide sequence homology between the human and rat proenkephalin gene reveals the presence of highly conserved regions within both the coding and noncoding portions of the genes. Enkephalin-coding sequences as well as 5' flanking sequences appear to be the most highly conserved. The importance and possible function of these sequences are discussed.     

Mutation Analysis of the 5′ Untranslated Region of the Cold Shock cspA mRNA of Escherichia coli

The mRNA for CspA, a major cold shock protein in Escherichia coli, contains an unusually long (159 bases) 5' untranslated region (5'-UTR), and its stability has been shown to play a major role in cold shock induction of CspA. The 5'-UTR of the cspA mRNA has a negative effect on its expression at 37 degrees C but has a positive effect upon cold shock. In this report, a series of cspA-lacZ fusions having a 26- to 32-base deletion in the 5'-UTR were constructed to examine the roles of specific regions within the 5'-UTR in cspA expression. It was found that none of the deletion mutations had significant effects on the stability of mRNA at both 37 and 15 degrees C. However, two mutations (Delta56-86 and Delta86-117) caused a substantial increase of beta-galactosidase activity at 37 degrees C, indicating that the deleted regions contain a negative cis element(s) for translation. A mutation (Delta2-27) deleting the highly conserved cold box sequence had little effect on cold shock induction of beta-galactosidase. Interestingly, three mutations (Delta28-55, Delta86-117, and Delta118-143) caused poor cold shock induction of beta-galactosidase. In particular, the Delta118-143 mutation reduced the translation efficiency of the cspA mRNA to less than 10% of that of the wild-type construct. The deleted region contains a 13-base sequence named upstream box (bases 123 to 135), which is highly conserved in cspA, cspB, cspG, and cspI, and is located 11 bases upstream of the Shine-Dalgarno (SD) sequence. The upstream box might be another cis element involved in translation efficiency of the cspA mRNA in addition to the SD sequence and the downstream box sequence. The relationship between the mRNA secondary structure and translation efficiency is discussed.     

Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA.

The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA (mtDNA) from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA. Both D-loop regions are bounded by the genes for tRNAPhe and tRNAPro. This region contains the most highly divergent sequences in mtDNA with the exceptions of three small conserved sequence blocks near the 5' ends of D-loop strands, a 225 nucleotide conserved sequence block in the center of the D-loop strand template region, and a short sequence associated with the 3' ends of D-loop strands. A sequence similar to that associated with the 3' termini of D-loop strands overlaps one of the conserved sequence blocks near the 5' ends of D-loop strands. The large, central conserved sequence probably does not code for a protein since no open reading frames are discretely conserved. Numerous symmetric sequences and potential secondary structures exist in these sequences, but none appear to be clearly conserved between species.     

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.     

Identification of GB virus C variants by phylogenetic analysis of 5'-untranslated and coding region sequences.

Phylogenetic analysis of 44 GB virus C (GBV-C) 5'-untranslated region (5'-UTR) sequences from 37 individuals suggested the presence of GBV-C genotypes (A. S. Muerhoff, J. N. Simons, T. P. Leary, J. C. Erker, M. L. Chalmers, T. J. Pilot-Matias, G. J. Dawson, S. M. Desai, and I. K. Mushahwar, J. Hepatol. 25:379-384, 1996) that correlated with geographic origin: type 1, 2a and 2b, and 3 isolates are found predominantly in West Africa, the United States and Europe, and Japan, respectively. We have extended our analysis to include 5'-UTR sequences from 129 globally distributed GBV-C isolates and sequences from the second envelope protein (E2) gene and nonstructural (NS) regions 3 and 5b from a subset of these isolates. Bootstrap analysis of a 157-nucleotide segment of the 5'-UTR from 129 sequences provided weak support for the existence of the four major groups of GBV-C isolates previously described, although phylogenetic analysis of a 374-nucleotide segment of the 5'-UTR from 83 isolates provided stronger support. Thus, the groups of GBV-C variants previously identified upon analysis of the entire 5'-UTR can be distinguished by analysis of the shorter, 374-nucleotide region from the 5'-UTR. In contrast, independent analysis of the E2, NS3, or NS5b region sequences does not identify groups of GBV-C variants that correlate with geographic origin. However, bootstrap analysis of these coding sequences, when linked to form colinear sequences, demonstrates that longer coding regions can produce GBV-C groupings that are similar to that determined from 5'-UTR sequence analysis. The inability to distinguish between GBV-C variants by using small segments of coding sequence suggests that the GBV-C genome is constrained. As a result of these constraints, there is a high degree of nucleotide and amino acid sequence conservation between isolates from widely separated geographic areas. Hence, substitutions at many nucleotide positions are not tolerated, so that substitutions at the positions which can change are saturated, thereby obscuring the evolutionary relationships.