An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3'' untranslated region of lymphokine mRNA.

Considerable evidence suggests that the metabolism of lymphokine mRNAs can be selectively regulated within the cytoplasm. However, little is known about the mechanism(s) that cells use to discriminate lymphokine mRNAs from other mRNAs within the cytoplasm. In this study we report a sequence-specific cytoplasmic factor (AU-B) that binds specifically to AUUUA multimers present in the 3' untranslated region of lymphokine mRNAs. AU-B does not bind to monomeric AUUUA motifs nor to other AU-rich sequences present in the 3' untranslated region of c-myc mRNA. AU-B RNA-binding activity is not present in quiescent T cells but is rapidly induced by stimulation of the T-cell receptor/CD3 complex. Induction of AU-B RNA-binding activity requires new RNA and protein synthesis. Stabilization of lymphokine mRNA induced by costimulation with phorbol myristate acetate correlates inversely with binding by AU-B. Together, these data suggest that AU-B is a cytoplasmic regulator of lymphokine mRNA metabolism.     

Synthesis of predominantly unspliced cytoplasmic RNAs by chimeric herpes simplex virus type 1 thymidine kinase-human beta-globin genes.

The herpes simplex virus type 1 thymidine kinase (tk) gene lacks introns and produces stable mRNA in the absence of splicing. We have prepared a hybrid gene by placing the first exon, first intron (first intervening sequence, designated IVS1), and most of the second exon of the normal human beta-globin gene into the 3' untranslated region of the tk gene. Although this hybrid gene contains all globin sequences presumed necessary for the splicing of IVS1, predominantly, unspliced stable cytoplasmic RNA is produced in both long- and short-term expression assays. Moreover, stable unspliced cytoplasmic RNA is detected whether the intron is situated in a sense or an antisense orientation. Efficient splicing of IVS1 is obtained either by deleting the majority of tk coding sequences or by relocating the globin sequences from the 3' to the 5' untranslated region of the tk gene.     

Mouse connexin 45: genomic cloning and exon usage

Connexin 45 is a gap junction protein that is prominent in early embryos and is widely expressed in many mature cell types. To elucidate its gene structure, expression, and regulation, we isolated mouse Cx45 genomic clones. Alignment of the genomic DNA and cDNA sequences revealed the presence of three exons and two introns. The first two exons contained only 5' untranslated sequences, while exon 3 contained the remaining 5' UTR, the entire coding region, and the 3' UTR. An RT-PCR with exon-specific primers was utilized to examine exon usage in F9 mouse embryonal carcinoma cells and adult mouse tissues. In all samples, PCR products amplified using exon 2/exon 3 or exon 3/exon 3 primer pairs were much more abundant than products produced using exon 1/exon 2 or exon 1/exon 3 primer pairs, suggesting that Cx45 mRNAs containing exon 1 were relatively rare compared with mRNAs containing the other exons. Rapid amplification of cDNA ends (5'-RACE) was performed using antisense primers from within exon 3 and template RNA prepared from F9 cells or from adult mouse kidney. We obtained multiple RACE products from both templates, including products that contained all three exons and were spliced identically to the cDNA. However, clones were also isolated (from kidney) that began within the region previously identified as intron 1 and continued upstream with a sequence identical to the cDNA, including splicing to exon 3. These results show that mouse Cx45 has a gene structure that differs from that of previously studied connexins and allows the production of heterogeneous Cx45 mRNAs with differing 5' UTRs. These differences might contribute to regulation of Cx45 protein levels by modulating mRNA stability or translational efficiency.     

Sequences that confer beta-tubulin autoregulation through modulated mRNA stability reside within exon 1 of a beta-tubulin mRNA

Synthesis of alpha- and beta-tubulin is controlled in animal cells by a novel autoregulatory mechanism: the concentration of unpolymerized subunits specifies the level of tubulin mRNAs. Using transient DNA transfection, we have localized the sequences that identify a beta-tubulin RNA as a substrate for autoregulation. Insertion of as few as 106 nucleotides (57 bases of 5' untranslated region and 49 coding nucleotides) from a beta-tubulin gene into a thymidine kinase gene is sufficient to make expression of the resultant chimeric RNA regulated as if it were an authentic beta-tubulin mRNA. Furthermore, all 5' untranslated region sequences can be deleted without disrupting regulation. We conclude that this novel autoregulatory pathway is specified by cytoplasmic events that modulate mRNA stability through sequences lying within the first 16 translated codons of a beta-tubulin mRNA.     

Transcriptional activation of lck by retrovirus promoter insertion between two lymphoid-specific promoters

p56lck, a member of the src family of cytoplasmic tyrosine protein kinases, is expressed primarily in lymphoid cells. Previous RNase protection data demonstrated the existence of at least two lck mRNAs (type I and type II) with different 5' untranslated regions in most T cells. These have been found here to arise from two separate promoters. S1 nuclease analysis and primer extension were used to locate the site of initiation of type I lck mRNA. The nucleotide sequence of the region upstream of this start site contains no classical promoter motifs. A cDNA clone of type II lck mRNA was isolated. The promoter of this mRNA must be more than 10 kilobases upstream of the type I promoter region. In two murine thymoma cell lines, LSTRA and Thy19, lck is expressed at elevated levels as a result of Moloney murine leukemia virus retrovirus promoter insertion. p56lck is encoded in these cells by a hybrid virus-lck mRNA containing the 5' untranslated region of Moloney virus mRNA. The structures and the sites of integration of the proviruses upstream of lck in these cells were examined by molecular cloning and Southern analysis. A truncated and rearranged provirus, flanked by 554 nucleotides (nt) of duplicated cellular sequences, was found 962 nt upstream of the start site for type I lck mRNA in LSTRA cells. What appears to be a Moloney mink cytopathic focus-forming provirus was found between 584 to 794 nt upstream of the start site for type I lck mRNA in Thy19 cells. Thus in both tumor cell lines, viral DNA is present between the promoters for type I and type II lck mRNAs. Comparison of the sequences of the 5' ends of the lck and c-src genes suggests that divergence of these two genes involved exon shuffling and that a homolog of the neuronal c-src(+) exon is not present in lck.