SR proteins ASF/SF2 and SRp55 participate in tissue factor biosynthesis in human monocytic cells.

BACKGROUND: Human monocytes express two naturally occurring forms of circulating tissue factor (TF) - full-length TF, a membrane-spanning protein, and alternatively spliced TF, a soluble molecule. Presence of the variable exon 5 in TF mRNA determines whether the encoded TF protein is transmembrane, or soluble. Recently, an essential SR protein ASF/SF2 was implicated in TF pre-mRNA processing in human platelets. OBJECTIVE: To examine molecular mechanisms governing regulated processing of TF pre-mRNA in human monocytic cells. METHODS AND RESULTS: In silico analysis of the human TF exon 5, present only in full-length TF mRNA, revealed putative binding motifs termed exonic splicing enhancers (ESE) for the SR proteins ASF/SF2 and SRp55, which were found to be abundantly expressed in monocytic cell lines THP-1 and SC, as well as monocyte-enriched peripheral blood mononuclear cells (PBMC). Using a splice competent mini-gene reporter system transiently expressed in monocytic cells, it was determined that weakening of either five closely positioned ASF/SF2 ESE (bases 87-117) or a single conserved SRp55 ESE (base 39) results in severe skipping of exon 5. ASF/SF2 and SRp55 were found to physically associate with the identified ESE. CONCLUSIONS: SR proteins ASF/SF2 and SRp55 appear to interact with the variable TF exon 5 through ESE at bases 39 and 87-117. Weakening of the above ESE modulates splicing of TF exon 5. This study is the first to identify and experimentally characterize cis-acting splicing elements involved in regulated biosynthesis of human TF.

Endoribonuclease RNase III is essential in Bacillus subtilis.

The rncS gene of Bacillus subtilis encodes Bs-RNase III, a narrow-specificity endoribonuclease. Previous attempts to disrupt rncS were unsuccessful. Here, a strain was constructed in which Bs-RNase III expression was dependent upon transcription of rncS from a temperature-sensitive plasmid. Growth of this strain at the non-permissive temperature resulted in 90-95% cell death, and virtually all the cells that survived retained the rncS-expressing plasmid. Thus, we conclude that rncS is essential in B. subtilis. The rncS conditional strain also revealed that Bs-RNase III participates in the processing of ribosomal RNA, in addition to processing small cytoplasmic RNA, a member of the signal recognition particle RNA family. Most significantly, a rare rncS null strain was isolated that will aid further study of the critical role Bs-RNase III plays in B. subtilis.

The yvaJ gene of Bacillus subtilis encodes a 3'-to-5' exoribonuclease and is not essential in a strain lacking polynucleotide phosphorylase.

Studies of Bacillus subtilis RNases that are involved in mRNA degradation reveal a different pattern from that of Escherichia coli. A strain lacking polynucleotide phosphorylase, the major 3'-to-5' exoribonuclease activity in cell extracts, is viable. Here, we show that the B. subtilis yvaJ gene encodes a second 3'-to-5' exoribonuclease. A strain lacking both of these RNases grows slowly but is viable. The existence of another, as yet unknown, 3'-to-5' exoribonuclease in B. subtilis is suggested.

Protection against 3'-to-5' RNA decay in Bacillus subtilis.

A 320-nucleotide RNA with several characteristic features was expressed in Bacillus subtilis to study RNA processing. The RNA consisted of a 5'-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3'-proximal end of the ermC transcriptional unit. Comparison of RNA processing in a wild-type strain and a strain in which the pnpA gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro. Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure. This structure did not provide a block to processivity in the pnpA strain, suggesting that it was specific for PNPase. An abundant RNA with a 3' end located in the ermC coding sequence was detected in the pnpA strain but not in the wild type, indicating that this block is specific for a different 3'-to-5' exonuclease. The finding of impediments to 3'-to-5' degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.

Decay of ermC mRNA in a polynucleotide phosphorylase mutant of Bacillus subtilis.

ermC mRNA decay was examined in a mutant of Bacillus subtilis that has a deleted pnpA gene (coding for polynucleotide phosphorylase). 5'-proximal RNA fragments less than 400 nucleotides in length were abundant in the pnpA strain but barely detectable in the wild type. On the other hand, the patterns of 3'-proximal RNA fragments were similar in the wild-type and pnpA strains. Northern blot analysis with different probes showed that the 5' end of the decay intermediates was the native ermC 5' end. For one prominent ermC RNA fragment, in particular, it was shown that formation of its 3' end was directly related to the presence of a stalled ribosome. 5'-proximal decay intermediates were also detected for transcripts encoded by the yybF gene. These results suggest that PNPase activity, which may be less sensitive to structures or sequences that block exonucleolytic decay, is required for efficient decay of specific mRNA fragments. However, it was shown that even PNPase activity could be blocked in vivo at a particular RNA structure.

Bacillus subtilis RNase III cleaves both 5'- and 3'-sites of the small cytoplasmic RNA precursor.

Bacillus subtilis small cytoplasmic RNA (scRNA) is a member of the signal recognition particle RNA family. It is transcribed as a 354-nucleotide primary transcript and processed to a 271-nucleotide mature scRNA. In the precursor, the 5'- and 3'-flanking regions form a stable double-stranded structure based on their complementary sequence. This structure is similar to those of substrates for the double-stranded RNA processing enzyme, RNase III. The B. subtilis enzyme that has similar activity to Escherichia coli RNase III has been purified and is designated Bs-RNase III. Recently, B. subtilis rncS has been shown to encode Bs-RNase III (Wang, W., and Bechhofer, D. H. (1997) J. Bacteriol. 179, 7379-7385). We show here that Bs-RNase III and the purified His-tagged product of rncS cleave pre-scRNA at both 5'- and 3'-sites to produce an intermediate scRNA (scRNA-275), although processing at the 3'-site is less efficient. The 5'-end of scRNA-275 was identical to that of the mature scRNA, whereas it contains four excess nucleotides at the 3'-end. Bs-RNase III cleavage yields a two-base 3'-overhang, which is consistent with the manner in which E. coli RNase III cleaves. We also show that truncation of the rncS gene affected processing, and significant amounts of an intermediate scRNA (scRNA-275) were found to accumulate in the rncS-truncated mutant. It is concluded that Bs-RNase III is an enzyme that processes pre-scRNA.

tetA(L) mutants of a tetracycline-sensitive strain of Bacillus subtilis with the polynucleotide phosphorylase gene deleted.

A Bacillus subtilis strain with the polynucleotide phosphorylase gene deleted was sensitive for growth in the presence of tetracycline. This strain was used to select for tetracycline-resistant mutants. A point mutation in the tetA(L) promoter and a spontaneously occurring tetA(L) gene copy number mutant were characterized.

Bacillus subtilis tetA(L) gene expression: evidence for regulation by translational reinitiation.

The tetA(L) gene of Bacillus subtilis encodes a transmembrane protein that can function as a Tc-metal/H+ antiporter, conferring low-level resistance to tetracycline. The TetA(L) coding sequence is preceded by a leader region that contains a 20-amino-acid open reading frame and an appropriately spaced ribosome binding site. Expression of the gene is induced by addition of tetracycline, which is thought to act by binding to ribosomes that translate the tetA(L) leader peptide coding sequence. Here we demonstrate that induction of tetA(L) expression includes minor transcriptional and major translational components. Deletion and point mutations of the tetA(L) leader region were constructed to probe the mechanism of translational induction. To account for the observed mutant phenotypes, we propose that tetA(L) expression is regulated by a translational reinitiation mechanism.

Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci.

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.

Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain.

Deletion of the tetA(L) chromosomal region of Bacillus subtilis in a strain designated JC112 increased the strain's sensitivity to low tetracycline concentrations. It also resulted in phenotypic changes that correlate with the previously found role of TetA(L) in mediating electrogenic NA+/H+ antiport. Growth of JC112 was impaired relative to that of the wild type at both pH 7.0 and 8.3; Na(+)- and K(+)-dependent pH homeostases were impaired at alkaline pH. The phenotype of JC112 was complemented by plasmid-borne tetA(L) and related tet(K) genes; the antiport activity conferred by the tet(K) gene had an apparently higher preference for K+ over Na+ than that conferred by tetA(L). The data were consistent with TetA(L) being the major Na+(K+)/H+ antiporter involved in pH homeostasis in B. subtilis as well as a significant Na+ extrusion system. The phenotype of JC112 was much more pronounced than that of an earlier transposition mutant, JC111, with a disruption in the putative tetA(L) promoter region. Northern (RNA) blot analysis of tetA(L) RNA from wild-type and JC111 strains revealed the same patterns. That JC111 nevertheless exhibited some Na+ and alkali sensitivity may be accounted for by disruption of regulatory features that, in the wild type, allow increased tetA(L) expression under specific conditions of pH and monovalent cation concentration. Evidence for several different regulatory effects emerged from studies of lacZ expression from the transposon of JC111 and from a tetA(L)-lacZ translational fusion introduced into the amyE locus of wild-type and JC112 strains.

Properties of a Bacillus subtilis polynucleotide phosphorylase deletion strain.

The pnpA gene of Bacillus subtilis, which codes for polynucleotide phosphorylase (PNPase), has been cloned and employed in the construction of pnpA deletion mutants. Growth defects of both B. subtilis and Escherichia coli PNPase-deficient strains were complemented with the cloned pnpA gene. RNA decay characteristics of the B. subtilis pnpA mutant were studied, including the in vivo decay of bulk mRNA and the in vitro decay of either poly(A) or total cellular RNA. The results showed that mRNA decay in the pnpA mutant is accomplished despite the absence of the major, Pi-dependent RNA decay activity of PNPase. In vitro experiments suggested that a previously identified, Mn2+ -dependent hydrolytic activity was important for decay in the pnpA mutant. In addition to a cold-sensitive-growth phenotype, the pnpA deletion mutant was found to be sensitive to growth in the presence of tetracycline, and this was due to an increased intracellular accumulation of the drug. The pnpA deletion strain also exhibited multiseptate, filamentous growth. It is hypothesized that defective processing of specific RNAs in the pnpA mutant results in these phenotypes.

In vitro processing activity of Bacillus subtilis polynucleotide phosphorylase.

A phosphate-dependent exonuclease activity was identified in purified protein fractions from Bacillus subtilis that were selected for binding to poly(I)-poly(C) agarose. Based on the characteristics of the degradation products and the absence of this activity in a pnpA strain, which contains a transposon insertion in the B. subtilis PNPase gene (Luttinger et al., 1996--accompanying paper), this exonuclease activity was shown to be due to polynucleotide phosphorylase (PNPase). Processive 3'-to-5' exonucleolytic degradation of an SP82 phage RNA substrate was stalled at a particular site. Structure probing of the RNA showed that the stall site was downstream of a particular stem-loop structure. A similar stall site was observed for an RNA that comprised the intergenic region between the B. subtilis rpsO and pnpA genes. The ability to initiate degradation of a substrate that had a stem structure at its 3' end differed for the B. subtilis and Escherichia coli PNPase enzymes.

A polypurine sequence that acts as a 5' mRNA stabilizer in Bacillus subtilis.

A segment of early RNA from Bacillus subtilis bacteriophage SP82 was shown to function as a 5' stabilizer in B. subtilis. Several heterologous RNA sequences were stabilized by the presence of the SP82 sequence at the 5' end, and expression of downstream coding sequences was increased severalfold. The SP82 RNA segment encodes a B. subtilis RNase III cleavage site, but cleavage by B. subtilis RNase III was not required for stabilization. The sequence that specifies 5' stabilizer function was localized to a polypurine sequence that resembles a ribosome binding site. The ability of the SP82 sequence to stabilize downstream RNA was dependent on its position relative to the 5' end of the RNA. These results demonstrate the existence of a new type of 5' stabilizer in B. subtilis and indicate that attack at the 5' end is a principal mechanism for initiation of mRNA decay in B. subtilis.

Substrate specificity of an RNase III-like activity from Bacillus subtilis.

Bacillus subtilis bacteriophage SP82 codes for several early RNAs that were shown previously to be cleaved by an RNase III-like enzyme called "Bs-RNase III." Cloning of DNA fragments that encode these RNA sequences downstream of a T7 RNA polymerase promoter allowed the synthesis of substrates that were used to test the cleavage specificity of Bs-RNase III, which was purified from a protease-deficient strain of B. subtilis. Single nucleotide changes at or near the cleavage site and deletions upstream and downstream of the cleavage site were constructed. The effects of these changes on the rate of Bs-RNase III cleavage were measured. The activity of Bs-RNase III and Escherichia coli RNase III on heterologous substrates was also tested. Although the local environment of the site of Bs-RNase III cleavage appears very similar to that of E. coli RNase III, there are important differences in their substrate specificity.

An intron in the thymidylate synthase gene of Bacillus bacteriophage beta 22: evidence for independent evolution of a gene, its group I intron, and the intron open reading frame.

The thymidylate synthase gene (thy) (EC 2.1.1.45) of Bacillus subtilis bacteriophage beta 22 has a self-splicing, group I intron inserted into a highly conserved region of the coding sequence. The intron is very similar to one that is inserted 21 bp further downstream in the homologous thymidylate synthase gene (td) of Escherichia coli bacteriophage T4. In contrast, the amino acid sequences of the bacteriophage thymidylate synthases are highly divergent. The beta 22 intron has a fragmentary open reading frame (ORF) that encodes a putative helix-turn-helix DNA-binding motif, similar to one at the carboxyl terminus of the homing endonuclease (I-TevI) encoded by the T4 td intron. The td ORF and the thy ORF fragments are inserted into different regions of their respective intron structures. These results suggest that the thymidylate synthase genes, their introns, and their respective intron-ORFs all have separate evolutionary histories and that the acquisition of the intron could not have occurred by a simple homing event.

Initiation of mRNA decay in Bacillus subtilis.

Ribosome stalling in the leader region of ermC mRNA results in a 10-15-fold increase in ermC mRNA half-life in Bacillus subtilis. Fusion of the ermC 5' regulatory region to several B. subtilis coding sequences resulted in induced stability of the fusion RNAs, showing that the ermC 5' region acts as a general '5' stabilizer'. RNA products of an ermC-lacZ transcriptional fusion were inducibly stable in the complete absence of translation and included a small RNA that is likely to be a decay product arising by blockage of a 3'-to-5' exoribonuclease activity. Insertion of sequences that encode endonucleolytic cleavage sites into the ermC coding sequence resulted in cleavage products whose stability depended on the nature of their 5' and 3' ends. It can be concluded from this study that initiation of mRNA decay in B. subtilis generally occurs at or near the 5' terminus.

Regulation of the macrolide-lincosamide-streptogramin B resistance gene ermD.

The erythromycin resistance gene ermD, which encodes an rRNA methylase protein, has an unusually long leader region (354 nucleotides). Previously, a single promoter-proximal leader peptide coding sequence was recognized from the nucleotide sequence, and erythromycin-induced ribosome stalling in this sequence was proposed to be required for the induction of methylase translation. We characterized spontaneously occurring and in vitro-constructed leader region mutations in an effort to understand the function of various segments of the long ermD leader region. A second leader peptide coding sequence was identified, and the location of insertion and point mutations that expressed ermD methylase constitutively suggested that translation of the second leader peptide is controlled by ribosome stalling in the first leader peptide. From Northern RNA blot analysis of ermD transcription, it appears that regulation of ermD expression is not by transcriptional attenuation.

Effect of ermC leader region mutations on induced mRNA stability.

Induction of translation of the ermC gene product in Bacillus subtilis occurs upon exposure to erythromycin and is a result of ribosome stalling in the ermC leader peptide coding sequence. Another result of ribosome stalling is stabilization of ermC mRNA. The effect of leader RNA secondary structure, methylase translation, and leader peptide translation on induced ermC mRNA stability was examined by constructing various mutations in the ermC leader region. Analysis of deletion mutations showed that ribosome stalling causes induction of ermC mRNA stability in the absence of methylase translation and ermC leader RNA secondary structure. Furthermore, deletions that removed much of the leader peptide coding sequence had no effect on induced ermC mRNA stability. A leader region mutation was constructed such that ribosome stalling occurred in a position upstream of the natural stall site, resulting in induced mRNA stability without induction of translation. This mutation was used to measure the effect of mRNA stabilization on ermC gene expression.

A method for sequencing polymerase chain reaction products can be used to sequence Bacillus subtilis "miniprep" plasmid DNA.

Several methods for sequencing double-stranded plasmid DNA isolated from E. coli have been described. These methods are usually not effective when used to sequence plasmid DNA isolated from Bacillus subtilis. In the course of developing a simplified version of a previously published protocol for polymerase chain reaction product sequencing, it was found that this protocol could be used for sequencing plasmid DNA isolated from Bacillus subtilis.

Triple post-transcriptional control.

The ermC gene confers resistance to MLS antibiotics in a Bacillus subtilis host. Synthesis of the ermC gene product, a ribosomal RNA methylase, is inducible by the addition of subinhibitory concentrations of erythromycin. Regulation of ermC gene expression occurs at the post-transcriptional level in three ways: translational attenuation, translational autoregulation, and messenger RNA stabilization.