Elimination of Introns at the Drosophila Suppressor-of-Forked Locus by P-Element-Mediated Gene Conversion Shows That an RNA Lacking a Stop Codon Is Dispensable

The suppressor of forked [su(f)] locus affects the phenotype of mutations caused by transposable element insertions at unlinked loci. It encodes a putative 84-kD protein with homology to two proteins involved in mRNA 3' end processing; the product of the yeast RNA14 gene and the 77-kD subunit of human cleavage stimulation factor. Three su(f) mRNAs are produced by alternative polyadenylation. The 2.6- and 2.9-kb mRNAs encode the same 84-kD protein while a 1.3-kb RNA, which terminates within the fourth intron, is unusual in having no stop codon. Using P-element-mediated gene replacement we have copied sequences from a transformation construct into the su(f) gene creating a su(f) allele at the normal genomic location that lacks the first five introns. This allele is viable and appears wild type for su(f) function, demonstrating that the 1.3-kb RNA and the sequences contained within the deleted introns are dispensable for su(f) function. Compared with studies on gene replacement at the white locus, chromosomal breaks at su(f) appear to be less efficiently repaired from ectopic sites, perhaps because of the location of su(f) at the euchromatin/heterochromatin boundary on the X chromosome.     

The human U1-70K snRNP protein: cDNA cloning, chromosomal localization, expression, alternative splicing and RNA-binding.

We have isolated and sequenced cDNA clones encoding the human U1-70K snRNP protein, and have mapped this locus (U1AP1) to human chromosome 19. The gene produces two size classes of RNA, a major 1.7-kb RNA and a minor 3.9-kb RNA. The 1.7-kb species appears to be the functional mRNA; the role of the 3.9-kb RNA, which extends further in the 5' direction, is unclear. The actual size of the hU1-70K protein is probably 52 kd, rather than 70 kd. The protein contains three regions similar to known nucleic acid-binding proteins, and it binds RNA in an in vitro assay. Comparison of the cDNA sequences indicates that there are multiple subclasses of mRNA that arise by alternative pre-mRNA splicing of at least four alternative exon segments. This suggests that multiple forms of the hU1-70K protein may exist, possibly with different functions in vivo.     

Characterization of new human c-myc mRNA species produced by alternative splicing

Characterization of two human c-myc cDNAs corresponding to the mRNAs 2.5 and 3.1 kb in length transcribed from PO previously demonstrated the existence of alternative acceptor sites at the end of intron 1 and intron 2, respectively [Bentley, D.L. and Groudine, M. (1986) Mol. Cell. Biol. 6, 3481–3489]. We investigated the use of these alternative acceptor sites in each c-myc mRNA species. We characterized cDNAs corresponding to c-myc mRNAs transcribed in the SW613-S human carcinoma cell line. The use of the alternative acceptor site at the end of intron I was demonstrated in two out of 10 cDNAs corresponding to the 3.1-kb mRNA transcribed from PO and in three out of 10 cDNAs corresponding to the mRNAs transcribed from P1 or P2. The use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from PO. The mRNAs resulting from the use of this acceptor site would encode for a variant form of the p67 polypeptide lacking one amino-acid residue. Conversely, the use of the alternative acceptor site at the end of intron 2 was not found in any of the cDNAs corresponding to the mRNAs transcribed from PO (0/10), from P1 or P2 (0/10) and from P3 (0/10). In the course of this study, we isolated a cDNA corresponding to another new c-myc mRNA species. This mRNA is produced by alternative splicing within intron 1 and encodes only for p64.     

Hypoxia down-regulates sFlt-1 (sVEGFR-1) expression in human microvascular endothelial cells by a mechanism involving mRNA alternative processing

sFlt-1 (soluble Flt-1) potently inhibits angiogenesis by binding extracellularly to VEGF (vascular endothelial growth factor). In the present paper, we report that hypoxia down-regulates sFlt-1 expression in HMVECs (human microvascular endothelial cells), a constituent of microvessels where angiogenesis occurs. Hypoxia (5-1% O?) increased VEGF expression in HMVECs. In contrast, the levels of sFlt-1 mRNA and protein in HMVECs decreased significantly as the O? concentration fell, whereas mFlt-1 (membrane-bound Flt-1) mRNA and protein remained unchanged. This suggested that hypoxia selectively regulates alternative 3'-end processing of sFlt-1 pre-mRNA. We have also demonstrated that sFlt-1 overexpression in lentiviral-construct-infected HMVECs counteracted VEGF-induced endothelial cell growth. We next identified cis-elements involved in sFlt-1 mRNA processing in HMVECs using a human Flt-1 minigene and found that two non-contiguous AUUAAA sequences function as the poly(A) signal. Furthermore, we identified a cis-element in intron 13 that regulates sFlt-1 mRNA processing. Mutagenesis of the U-rich region in intron 13 caused a significant decrease in the soluble-form/membrane-form RNA ratio in the minigene-transfected HMVECs. These results suggest that decreased sFlt-1 expression due to hypoxia contributes to hypoxia-induced angiogenesis and reveals a novel mechanism regulating angiogenesis by alternative mRNA 3'-end processing.     

Rapid phosphorylation of the L-myc protein induced by phorbol ester tumor promoters and serum.

We have examined post-translational modification of the L-myc protein using polyclonal and monoclonal antibodies against a peptide well conserved in the predicted amino acid sequences of the c-myc, N-myc and L-myc genes. These antibodies precipitate three polypeptides of Mr 60-66,000 from [35S]methionine or [32P]orthophosphate-labelled human small cell lung cancer cell lines expressing amplified L-myc genes, but not the other myc genes. Treatment of the L-myc immunoprecipitates with alkaline phosphatase prior to electrophoresis converts the three methionine-labelled polypeptides into a single band migrating at Mr 59,000, and efficiently removes radioactivity from the 32P-labelled L-myc protein, suggesting that, in contrast to the c-myc and N-myc proteins, the L-myc polypeptide heterogeneity is due to differential phosphorylation of a common precursor. When the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or serum is added to cultures of U-1690 cells the Mr 66,000 polypeptide is rapidly enriched while the Mr 60,000 form is decreased in the L-myc immunoprecipitates. This effect is correlated with the ability of phorbol ester and diacylglycerol analogues to activate protein kinase C. The TPA-induced phosphorylation of the L-myc protein occurs in a protein synthesis-independent manner as it is not inhibited by cycloheximide or anisomycin. These data indicate that the phosphorylation of the L-myc nuclear oncoprotein is modulated in response to TPA via a rapid signal transduction system involving protein kinase C. This mechanism could play an important role in the response of lung cells to e.g. bombesin-related growth factors.     

Fetal and variant alpha-fetoproteins are encoded by mRNAs that differ in sequence at the 5' end

The temperature-sensitive RLA209-15 fetal rat hepatocyte line grown at the nonpermissive temperature (40 degrees C, normal phenotype) produces authentic rat alpha-fetoproteins (AFPs) of 69K and 73K (fetal AFPs) which are encoded by a 2.2-kb mRNA. These cells also produce low levels of a 1.7-kb AFP mRNA and a 65K variant AFP when grown at the permissive temperature (33 degrees C, transformed phenotype). Hybrid-selected translation demonstrates that the 1.7-kb AFP mRNA encodes the 65K variant AFP. Northern blot hybridization and S1 nuclease analyses indicate that the 1.7-kb mRNA lacks sequences present in the first seven 5' exons of the 2.2-kb AFP mRNA. However, the 1.7- and 2.2-kb AFP mRNAs share common sequences extending from the beginning of the eighth exon (corresponding to nucleotide 873 of the fetal AFP mRNA) to the 3' end. Primer extension analysis suggests that the 1.7-kb RNA contains additional sequences 5' to the common regions shared by both AFP mRNAs. We have previously shown that adult rat liver produces a 1.7-kb AFP mRNA; we now report the isolation of a cDNA (ARFP5) encoding this variant AFP mRNA from an adult rat liver cDNA library. Restriction endonuclease mapping and sequence analysis of ARFP5 confirm that the 1.7- and 2.2-kb AFP mRNAs share similar sequences at the 3' region (approximately 1.1 kb). However, ARFP5 contains an additional 90 bp variant AFP mRNA-specific 5' sequence which is located in the seventh intron of the rat AFP gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of enzymatically active replication complexes from feline calicivirus-infected cells

A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative "3A-like" protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication.