TGTG,G clustering and Other Signals Near Non-Mammalian Vertebrate mRNA 3′ Termini: Some Implications

Abstract

Analysis of non-mammalian vertebrate DNA sequences in the 500 nucleotides preceding and 200 nucleotides following mRNA 3′ termini yields some distinct signals. In addition to the well known AATAAA sequence, TGTG recurs very frequently further downstream. GGGG, TGGG, GGAG and GAGG are frequent in this region too. It is suggested that unlike the AATAAA mRNA cleavage/processing signal, the other signals noted above are signals on the DNA, i.e. they are signals for mRNA termination. An asymmetric distribution of some complementary sequences, e.g. TGTG vs. CACA, GGGG vs. CCCC, on the same DNA strand is noted as well. A few other signals are also observed.     

Polyoma virus. The early region and its T-antigens.

The DNA sequence of the early coding region of polyoma virus is presented. It consists of 2739 nucleotides. The sequence predicts that more than one reading frame can be used to code for the three known polyoma virus early proteins (designated small, middle and large T-antigens). From the DNA sequence, the 'splicing' signals used in the processing of viral RNA to functional messenger RNAs can be predicted, as well as the sizes and sequences of the three proteins. Other unusual aspects of the DNA sequence are noted. Comparisons are made between the DNA sequences and the predicted amino acid sequences of the respective large T-antigens of polyoma virus and the related virus Simian Virus (SV) 40.     

Nucleotide sequences from the 3''-ends of vesicular stomatitis virus mRNA''s as determined from cloned DNA.

Molecular clones of vesicular stomatitis virus mRNA's were used to determine the 3'-terminal sequences of mRNA's encoding the N and NS proteins. This new approach to VSV mRNA sequencing allowed the first comparison of 3'-terminal sequences. The sequences showed a tetranucleotide homology, UAUG, immediately preceding the polyadenylic acid. In addition, both mRNA's had an AU-rich region including the tetranucleotide AUAU at positions 16 to 19 nucleotides from the polyadenylic acid. A possible secondary structure between the 3' end of N mRNA and the 5' end of the adjacent NS mRNA is noted. These structural features may serve as signals for termination (or cleavage) and polyadenylation of vesicular stomatitis virus mRNA's. Neither mRNA had the polyadenylic acidproximal hexanucleotide, AAUAAA, found in eucaryotic cellular and viral mRNA's transcribed from nuclear DNA. The probable location of the translation termination codon for the NS protein is only six nucleotides from polyadenylic acid in NS mRNA.     

Mobile dispersed genetic element MDG1 of Drosophila melanogaster: nucleotide sequence of long terminal repeats.

Long terminal repeats (LTRs) of two members of mdg1 family were sequenced. In the both cases, they are represented by perfect direct repeats 442 and 444 bp in length. Sixteen nucleotides in the LTRs of two different mdg1 elements are different. Each LTR contains slightly mismatched 16-nucleotide inverted repeats located at the ends of the LTR. Six base pairs closest to the termini of LTR form perfect inverted repeats. On the gene-distal sides of LTRs, short 4-nucleotide direct repeats are located, probably representing the duplication of a target DNA sequence arising from insertion of mdg. They are different in the two cases analyzed. Just as the other analyzed eukaryotic transposable elements, mdg1 starts with TGT and ends with ACA. Within the both strands of LTR, the sequences similar to Hogness box (a putative signal for RNA initiation, or a selector) and AATAAA blocks (putative polyadenylation signals) are present. The LTR of mdg1 contains many short direct and inverted repetitive sequences. These include a 10-nucleotide sequence forming a perfect direct repeat with the first ten nucleotides of the LTR. A region of LTR about 70 bp long is represented by simple repetitive sequences (TAT).     

The actin gene in yeast Saccharomyces cerevisiae: 5'' and 3'' end mapping, flanking and putative regulatory sequences

The 5' and 3' flanking regions of the yeast actin gene have been sequenced and the ends of the actin mRNA were determined by the single-strand nuclease mapping procedure. The mRNA starts with a pyrimidine residue 141 (or 140) nucleotides upstream from the initiation codon. The actin gene lacks a typical "TATA" box 30 base pairs upstream from the mRNA start site but it contains a region homologous to the canonical sequence 5'-GGCTCAATCT-3' which is found in several eukaryotic genes 70 to 80 bp upstream from the mRNA cap site. Judging from the S1 nuclease mapping, there are two populations of actin mRNA terminating 98 and 107 nucleotides downstream from the stop codon. The 3' termini are preceded by three AATAAA sequences found in most eukaryotic polyadenylated mRNAs.     

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.     

Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis.

In animal and viral pre-mRNAS, the process of polyadenylation is mediated through several cis-acting poly (A) signals present upstream and downstream from poly (A) sites. The situation regarding polyadenylation of higher plant pre-mRNAS, however, has remained obscure so far. In this paper, a search for putative poly (A) signals is made by considering the published data from 46 plant genomic DNA sequences. Certain domains in the 3' untranslated regions from nuclear genes of higher plants were compiled and occurrence of sequence motifs such as AATAAA, CAYTG, YGTGTTYY and YAYTG was scored in relation to poly (A) sites. Moreover, consensus sequences for important regions in the 3' untranslated sequences and poly (A) signals were also deduced from the data. It was inferred that sequence motifs similar to poly (A) signals exist around poly (A) sites but some of them are in entirely different spatial relationship than observed in other eukaryotes. This indicates their probable non-involvement in the process of polyadenylation in higher plants necessitating a functional analysis approach to define the plant specific poly (A) signals.     

Spatial constraints on polyadenylation signal function

Efficient cleavage and polyadenylation of eukaryotic messenger RNAs require at least two signal elements: an AAUAAA or closely related sequence located 7-30 base pairs (bp) upstream of the site of processing, and a G/U- or U-rich sequence located 3' to the cleavage site. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT-rich region. We have shown that the first AATAAA and the GT-rich region are essential for efficient processing, both in vivo and in vitro, whereas the second AATAAA does not appear to play any role in the formation of tk mRNA 3' ends. The failure of a signal containing only the second AATAAA and the GT-rich element to signal cleavage and polyadenylation suggested that these two elements might be too close together to constitute a functional polyadenylation signal. The experiments described in this report were directed at determining the effects on mRNA 3' end formation of alterations in spacing between signal elements. Wild-type tk contains 19 bp between these two elements. Constructs were made in which an AATAAA and the GT-rich region were separated by various distances ranging from 7 to 43 bp. The quantity and location of 3' ends of the tk mRNA produced by these constructs in Cos-1 cells were measured by S1 nuclease protection analysis. Signal efficiency was gradually reduced as the separation between the two signal elements was increased; with a separation of 43 bp, the signal functioned at approximately one-eighth the efficiency of the parental construction. Bringing the two signals closer together resulted in decreased signal efficiency; with a separation of 7 or 9 bp, no tk mRNA polyadenylated within the normal region was produced. Altering the sequences between these two elements without changing the distance had small effects on processing efficiency.     

The nucleotide sequence of rabbit embryonic globin gene beta 3

The nucleotide sequence of a rabbit embryonic globin gene, beta 3, has been determined from 161 base pairs (bp) on the 5' side of the mRNA cap site to 209 base pairs beyond the 3' poly A addition site. The 5' and 3' ends of mRNA from both embryonic globin genes beta 3 and beta 4 have been determined by an S1 protection assay. Sequences that are highly conserved in the 5' flanking region of eukaryotic structural genes, AATAAAA and CCAAT, are located -25 to -31 nucleotides and -81 to -85 nucleotides, respectively, before the cap site. The CCAAT sequence is duplicated at -108 to -112 nucleotides, as it is in the human fetal gamma-globin genes. Small (124 bp) and large (817 bp) intervening sequences are located between codons 30 and 31 and between 104 and 105, respectively. The sequence AATAAA precedes the predominant poly(A) addition site by 19 nucleotides. Although rabbit globin gene beta 3 is transcribed and translated almost exclusively in embryonic erythrocytes, it shares striking homology with the human gamma-globin genes which are expressed in erythrocytes from fetal liver. The evolutionary conservation of rabbit beta 3 and human gamma correlates well with their similar chromosomal positions in the two genes families.