Statistical analysis of the 5' untranslated region of human mRNA using "Oligo-Capped" cDNA libraries

We constructed 34 types of human "full-length enriched" and "5'-end enriched" cDNA libraries based on the "Oligo-Capping" method. We randomly picked and sequenced 10,000 clones from these libraries. BLAST analysis showed that about 50% of the cDNAs were identical to known genes. Among them, we selected 954 species of cDNA that should represent the entire sequence from the mRNA start sites. Compared with previously reported sequences, they were on average 45 bp longer in the 5'-end. Using these cDNA data, we statistically analyzed the sequence features of the 5'UTR. The average length of the 5'UTR was 125 bp, and there was little correlation with the corresponding mRNA length (correlation coefficient = 0.26). Of the 954 species of 5'UTR, 459 contained no in-frame terminator codon, which is against the common belief. Two hundred seventy-eight species contained at least one ATG codon upstream of the initiator ATG codon. We identified 569 upstream ATGs, in total, 63% of which adequately satisfied Kozak's criteria. These findings are contrary to the typical translation initiation model, which states that translation is initiated from the "first" ATG codon.     

Complete nucleotide sequence of the 5' noncoding region of human alpha-and beta-globin mRNA

The 5' noncoding regions of human alpha-and beta-globin mRNAs, 37 and 50 nucleotides in length, have been sequenced. A variation of the "plus and minus" gel technique described by Brownlee and Cartwright (1977) was used, and the results were cross-checked by the Maxam and Gilbert (1977) procedure. These studies completed the knowledge of all the noncoding region sequences of both mRNAs, and it was then possible to calculate their exact size. Human alpha-and beta-globin mRNAs are 575 and 626 nucleotides in length, excluding the poly(A). Furthermore, because the coding and 3' noncoding regions of the latter were known from previous studies (Marotta et al., 1977; Proudfoot, 1977), the primary structure of human beta-globin mRNA is now complete except for six ambiguities in the coding region. The human and rabbit 5' noncoding region sequences are about 80% homologous. This suggests that they are under a moderate selective pressure.     

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.     

cDNA cloning of human hnRNP protein A1 reveals the existence of multiple mRNA isoforms.

Protein A1 is one of the major component of mammalian ribonucleoprotein particles (hnRNP). Human protein A1 cDNA cloning and sequencing revealed the existence of at least two protein isoforms. Among the cDNAs examined, sequence differences were found both in the structural portion, leading to aminoacid changes (Tyr to Phe or Arg to Lys) and in the non translated 3'-region where two T-stretches of different length were observed. Interestingly one of the aminoacid substitutions falls into a consensus sequence common to many RNA binding proteins. Northern blot analysis of poly A+ RNAs from five human tissues revealed two mRNA forms of 1500 and 1900 n due to alternative polyadenylation. Analysis of genomic DNA showed at least 30 A1-specific sequences, some of which correspond to processed pseudogenes. These results suggest that protein A1 is encoded by a multigene family.     

Sequence of human DNA polymerase beta mRNA obtained through cDNA cloning

A cDNA library from polyA+ RNA of a human teratocarcinoma cell line in phage lambda gt11 was screened with a fragment of the rat beta-polymerase cDNA, lambda pol beta-10, as probe. Five positive phage were identified and plaque purified. The cDNA of one positive clone selected for detailed study was 1257 bp. This insert was sequenced and found to contain the coding region for beta-polymerase, as well as 163 bp and 137 bp from the 5' and 3' untranslated regions, respectively. The primary structure of human beta-polymerase (318 amino acids, Mr = 36, 133) deduced from the cDNA was similar to rat beta-polymerase (95% matched residues). The greatest difference between the sequences of the human and rat cDNAs was in the 3' untranslated regions (64% matched base residues). These results provide necessary sequence information for study of the human beta-polymerase gene.     

The AU-rich 3' untranslated region of human MDR1 mRNA is an inefficient mRNA destabilizer.

The human multidrug resistance gene MDR1 encodes a membrane-bound protein, referred to as P-glycoprotein, that acts as a pump to extrude toxins from cells. The 3' untranslated region (3'UTR) of the human MDR1 mRNA is very AU-rich (70%) and contains AU-rich sequences similar to those shown to confer rapid decay on c-myc, c-fos, and lymphokine mRNAs. We tested the ability of the MDR1 3'UTR to act as an mRNA destabilizing element in the human hepatoma cell line HepG2. The MDR1 mRNA has an intermediate half-life of 8 h in HepG2 cells compared to a half-life of 30 min for c-myc mRNA. The MDR1 mRNA half-life was prolonged to >20 h upon treatment with the protein synthesis inhibitor cycloheximide. We constructed expression vectors containing the human beta-globin coding region with the 3'UTR from either MDR1 or c-myc. The c-myc 3'UTR increased the decay of the chimeric mRNA, but the MDR1 3'UTR had no effect. We tested the ability of MDR1 3'UTR sequences to compete for interaction with AU-binding proteins in cell extracts; MDR1 RNA probes had a fivefold lower affinity for AU-binding proteins that interact with the c-myc AU-rich 3'UTR. Overall, our data suggest that the MDR1 3'UTR does not behave as an active destabilizing element in HepG2 cells.