Functional Analysis of Sequence Motifs Involved in the Polyadenylation of Trichomonas vaginalis mRNAs

ABSTRACTSynthesis of functional mRNA in eukaryotes involves processing of precursor transcripts, including the addition of a poly(A) tail at the 3′ end. A multiprotein complex recognizes a polyadenylation signal, generally the hexanucleotide AAUAAA in metazoans, to direct processing of the pre-mRNA. Based on sequence analysis of several cDNAs, we have previously suggested that the UAAA tetranucleotide (which may include the UAA translation stop codon) could be the polyadenylation signal inTrichomonas vaginalis, a parasitic protozoon that causes human trichomoniasis. This proposal is analyzed here with the aid of a transient-expression system of a reporter gene (catflanked byT. vaginalisactin noncoding sequences). When cells were transfected with a plasmid bearing the original 3′ untranslated region (UTR) sequence containing the UAAA motif, the resultingcatmRNA was polyadenylated similarly to the endogenous actin mRNA. Base changes in the UAAA sequence produced alterations to the polyadenylation site of the reporter mRNAs, while nucleotide substitutions at either side of UAAA did not. Furthermore, relocation of the UAAA motif redirected the processing and polyadenylation of the reporter mRNA. In addition, a pre-mRNA cleavage site for polyadenylation was defined. Interaction ofT. vaginalisproteins with the UAAA motif was shown by electrophoretic mobility shift assays. Based on our findings, we provide evidence that inT. vaginalisthe UAAA tetranucleotide has a role equivalent to that of the metazoan consensus AAUAAA polyadenylation signal.

The human cytomegalovirus UL78 gene is highly conserved among clinical isolates, but is dispensable for replication in fibroblasts and a renal artery organ-culture system

The human cytomegalovirus (HCMV) UL78 ORF is considered to encode a seven-transmembrane receptor. However, neither the gene nor the UL78 protein has been characterized so far. The objective of this study was to investigate the UL78 gene and to clarify whether it is essential for replication. UL78 transcription was activated early after infection, was inhibited by cycloheximide but not by phosphonoacetic acid, and resulted in a 1·7 kb mRNA. Later in the replication cycle, a second mRNA of 4 kb evolved, comprising the UL77 and UL78 ORFs. The 5′ end of the UL78 mRNA initiated 48 bp upstream of the translation start and the polyadenylated tail started 268 bp downstream of the UL78 translation stop codon within the UL79 ORF. By using bacterial artificial chromosome technology, a recombinant HCMV lacking most of the UL78 coding region was constructed. Successful reconstitution of the UL78-deficient virus proved that the gene was not essential for virus replication in fibroblasts. The deletion also did not reduce virus replication in ex vivo-cultured sections of human renal arteries. Analysis of viral proteins at different stages of the replication cycle confirmed these results. Among clinical HCMV isolates, the predicted UL78 protein was highly conserved. However, an accumulation of different single mutations could be found in the N-terminal region and at the very end of the C terminus. Due to the absence of an in vivo HCMV model, the role of UL78 in the pathogenesis of HCMV infection in humans remains unclear.

Identification of New Sequence Variants in the Leptin Gene

The leptin gene (LEP) has been linked to extreme obesity. However, no common obesity-related gene variants have been found to exist in the LEP. The present study was designed to investigate the LEP for variants by screening both the putative promoter and the coding region of this gene in obese Finnish subjects (n = 200; body mass index, >27 kg/m2). PCR-amplified DNA samples were subjected to single strand conformation analysis. A G144A substitution in codon 48 and a G328A substitution in codon 110 were identified in two obese subjects, both of whom had very low serum leptin levels. A rare silent C538T polymorphism was detected 33 bp downstream of the translation stop codon (TGA). A common polymorphism A19G was identified in the untranslated exon 1. This polymorphism was not associated with traits of obesity; in agreement, the allele frequencies were similar between 64 normal weight and 141 obese Finns. In summary, this study failed to find a common gene variant in the LEP associated with obesity, but introduces 2 rare mutations associated with very low serum leptin concentrations in 2 obese subjects.

Nucleotide Structure and Characterization of the Murine Gene Encoding Anticoagulant Protein C

SummaryThe 15,160 bp murine gene encoding anticoagulation protein C (PC) was cloned and sequenced, including 414 bp upstream of exon 1 and 80 bp downstream of the translation stop codon. Nine exons and eight introns were identified. The first exon was untranslated and contained the major transcriptional start site, the surrounding nucleotide sequence of which matched reasonably well with the consensus eukaryotic Cap element sequence. The translational initiator methio-nine residue was located in exon 2. The other introns were positioned as splices between the major domain units of the protein. The 5’ untranslated region contained two possible CCAAT sequences and GC boxes, but no TATA box was obvious within the optimal range of distances from the transcription start site. The 3’-flanking nucleotides included a probable polyadenylation site (ATTAAA), beginning 80 nucleotides downstream of the translation stop codon, and a downstream consensus sequence (AGTGTTTC) required for the efficient formation of a 3’ terminus of mRNA. Several high probability transcription factor recognition sequences, including proteins that are enriched in, or specific to, the liver, such as C/EBP, C/EBP, HNF1, and HNF3, have been located in the 5’ region of the gene. These results indicate that all elements are present for liver-based transcription of the gene for murine PC.

Identification of molecular defects in a subject with type I CD36 deficiency

We performed a molecular analysis of a subject whose platelets and monocytes did not express any cell surface CD36 (designated as a type I CD36 deficiency). Amplification of the 5′ half of platelet and monocyte CD36cDNA (corresponding to nucleotide [nt] 191–1009 of the published CD36 cDNA sequence [Oquendo et al, Cell, 58:95, 1989]) showed that two different-sized CD36 cDNAs existed. One cDNA was of predicted normal size, whereas the other was about 150 bp smaller than that predicted for normal CD36 cDNA. Amplification of the 3′ region of CD36 cDNA (nt 962–1714) in this subject showed only normal-sized CD36 cDNA. Cloning and nt sequence analysis of the cDNAs showed that the smaller sized CD36 cDNA had 161-bp deletion (from nt 331 to 491), and a dinucleotide deletion starting at nt position 539. The same dinucleotide deletion was also detected in the normal sized CD36 cDNA. Both deletions caused a frameshift leading to the appearance of a translation stop codon. RNA blot analysis and quantitative assay using the reverse transcription- polymerase chain reaction (RT-PCR) showed that the CD36 transcripts in both platelets and monocytes were greatly reduced. Comparison of the determined cDNA sequences with the genomic DNA sequence for the human CD36 gene showed that the dinucleotide deletion was located in exon 5, and that the 161-bp deletion corresponded to a loss of exon 4. PCR- based analysis using genomic DNA showed that this subject was homozygous for the dinucleotide deletion in exon 5. Except for the dinucleotide deletion, we could not find any abnormalities around exon 3, 4, and 5 including the splice junctions. These results suggested that the deletions in CD36 mRNA were likely to be responsible for instability of the transcripts, and the dinucleotide deletion in exon 5 might affect the splicing of exon 4.

Identification of molecular defects in a subject with type I CD36 deficiency

We performed a molecular analysis of a subject whose platelets and monocytes did not express any cell surface CD36 (designated as a type I CD36 deficiency). Amplification of the 5′ half of platelet and monocyte CD36cDNA (corresponding to nucleotide [nt] 191–1009 of the published CD36 cDNA sequence [Oquendo et al, Cell, 58:95, 1989]) showed that two different-sized CD36 cDNAs existed. One cDNA was of predicted normal size, whereas the other was about 150 bp smaller than that predicted for normal CD36 cDNA. Amplification of the 3′ region of CD36 cDNA (nt 962–1714) in this subject showed only normal-sized CD36 cDNA. Cloning and nt sequence analysis of the cDNAs showed that the smaller sized CD36 cDNA had 161-bp deletion (from nt 331 to 491), and a dinucleotide deletion starting at nt position 539. The same dinucleotide deletion was also detected in the normal sized CD36 cDNA. Both deletions caused a frameshift leading to the appearance of a translation stop codon. RNA blot analysis and quantitative assay using the reverse transcription- polymerase chain reaction (RT-PCR) showed that the CD36 transcripts in both platelets and monocytes were greatly reduced. Comparison of the determined cDNA sequences with the genomic DNA sequence for the human CD36 gene showed that the dinucleotide deletion was located in exon 5, and that the 161-bp deletion corresponded to a loss of exon 4. PCR- based analysis using genomic DNA showed that this subject was homozygous for the dinucleotide deletion in exon 5. Except for the dinucleotide deletion, we could not find any abnormalities around exon 3, 4, and 5 including the splice junctions. These results suggested that the deletions in CD36 mRNA were likely to be responsible for instability of the transcripts, and the dinucleotide deletion in exon 5 might affect the splicing of exon 4.

Postinduction turnoff of beta-interferon gene expression.

Viral induction of the human beta-interferon (IFN-beta) gene leads to a transient accumulation of high levels of IFN-beta mRNA. Previous studies have shown that the increase in IFN-beta mRNA levels after induction is due to an increase in the rate of IFN-beta gene transcription. In this paper, we show that the rapid postinduction decrease in the level of IFN-beta mRNA is due to a combination of transcriptional repression and rapid turnover of the mRNA. This transcriptional repression can be blocked with cycloheximide, suggesting that the synthesis of a virus-inducible repressor is necessary for the postinduction turnoff of the IFN-beta gene. Analysis of the sequence requirements for IFN-beta mRNA instability revealed two regions capable of destabilizing a heterologous mRNA. One destabilizer is an AU-rich sequence in the 3' untranslated region, and the other is located 5' to the translation stop codon.

Postinduction turnoff of beta-interferon gene expression

Viral induction of the human beta-interferon (IFN-beta) gene leads to a transient accumulation of high levels of IFN-beta mRNA. Previous studies have shown that the increase in IFN-beta mRNA levels after induction is due to an increase in the rate of IFN-beta gene transcription. In this paper, we show that the rapid postinduction decrease in the level of IFN-beta mRNA is due to a combination of transcriptional repression and rapid turnover of the mRNA. This transcriptional repression can be blocked with cycloheximide, suggesting that the synthesis of a virus-inducible repressor is necessary for the postinduction turnoff of the IFN-beta gene. Analysis of the sequence requirements for IFN-beta mRNA instability revealed two regions capable of destabilizing a heterologous mRNA. One destabilizer is an AU-rich sequence in the 3' untranslated region, and the other is located 5' to the translation stop codon.