Ribonucleic Acid Splicing Research Articles

Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein

Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein

Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein.

PTH-related protein (PTHrP) is the principle mediator of the syndrome of humoral hypercalcemia of malignancy and has potential paracrine actions on smooth muscle, epithelial cell growth, and placental calcium transport. The human PTHrP gene is complex: a combination of three promoters, one 5' alternative splicing event and alternative 3' splicing, which produces three PTHrP isoforms (139, 141, or 173 amino acids), results in multiple PTHrP messenger RNA (mRNA) species. We employed the RT-PCR technique to identify promoter usage and splicing patterns in a range of human cell lines. Cell line-specific utilization of the promoters and the 3' alternative splicing pathways was detected among bone, breast, kidney, and lung cell lines, although each cell line could potentially produce the three PTHrP isoforms. We also determined whether some of the known regulators of PTHrP differentially modulate promoter usage or splicing patterns. Dexamethasone decreased the abundance of each of the alternative mRNA species. In contrast, epidermal growth factor and transforming growth factor-beta treatment increased the abundance of each PTHrP mRNA species, with particularly marked effects on promoter 1- and promoter 2-initiated transcripts, especially those containing exon VII or VIII. Epidermal growth factor treatment was found to alter PTHrP splicing patterns in a manner consistent with increased transcription from promoters 1 and 2 and stabilization of exon VII- and IX-containing transcripts.

Human myometrial G alpha s-small (with serine) and Gs-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G alpha s protein isoforms in pregnancy and their down-regulation during labor.

In previous studies using specific G alpha s antibodies we have identified several human myometrial G alpha s protein isoforms with molecular masses of 45, 46, 47, 54, and 58 kDa, respectively. During pregnancy, levels of the 46- and 54-kDa proteins are significantly increased compared to those in nonpregnant myometrium and then decreased at the onset of labor. In this study we investigated the expression of G alpha s messenger ribonucleic acid (mRNA) splice variants, which are generated as a result of alternative splicing of a single mRNA precursor, in term pregnancy and parturition to determine whether there was any correlation with the observed changes in G alpha s protein isoforms. A myometrial G alpha s complementary DNA was synthesized using RT-PCR and cloned into pCRtmII suitable for preparation of riboprobes for use in ribonuclease protection assays. Using this technique, we identified at least three myometrial G alpha s mRNAs, including two forms of G alpha s-Large (with or without the serine at amino acid 87) and one form of G alpha s-Small (with the serine at amino acid 72). G alpha s Small (with the serine) and G alpha s-Large (with the serine) mRNAs encode for the 46- and 54-kDa G alpha s protein isoforms, respectively, and were increased in term pregnancy and then subsequently decreased after the onset of labor. Our data suggest that posttranscriptional regulation of G alpha s mRNAs may be important in the differential expression of G alpha s protein isoforms during pregnancy and labor.

Human myometrial G alpha s-small (with serine) and Gs-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G alpha s protein isoforms in pregnancy and their down-regulation during labor

Human myometrial G alpha s-small (with serine) and Gs-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G alpha s protein isoforms in pregnancy and their down-regulation during labor

Alternative RNA splicing of the hyaluronic acid receptor CD44 in the normal human brain and in brain tumors.

The cell-surface receptor for hyaluronic acid, CD44, is expressed by both normal and malignant cells. Numerous CD44 isoforms have recently been identified that are derived by alternative ribonucleic acid splicing. The expression of some CD44 isoforms has been shown to be involved in tumor progression and metastatic spread in a rat carcinoma model and in human carcinomas. In the present study, CD44 isoform expression was evaluated by reverse transcriptase-polymerase chain reaction (PCR) analysis in frozen sections derived from three samples of normal brain tissue and from 40 brain tumors, including samples of glioblastoma multiforme, anaplastic astrocytoma, low-grade astrocytoma, cerebral primitive neuroectodermal tumor, medulloblastoma, metastatic colon carcinoma, and metastatic melanoma. Normal brain tissue adjacent to the tumors was also examined in 14 of 18 glioblastomas. In all normal brain and tumor samples, with the exception of metastases from colon carcinoma, PCR analysis demonstrated one prominent product that corresponded to the CD44H hematopoietic form of CD44. Metastases from colon carcinoma demonstrated two prominent PCR amplification products corresponding to CD44H and CD44R1. These results suggest that CD44H is the predominant isoform of this protein in normal human brain tissue and in human neuroectodermal tumors of varying degrees of malignancy. The ability of CD44H to mediate tumor cell motility and invasiveness (in contrast to CD44R1) suggests that the CD44 alternative splicing pattern of neuroectoderm-derived tumors may enhance their local biological aggressiveness and intracerebral spread. The lack of expression of larger molecular weight CD44 variants by primary brain tumors may also partially explain why these tumors rarely metastasize to distant sites.

Relationship between GsαMessenger Ribonucleic Acid Splice Variants and the Molecular Forms of Gsa Protein in Rat Brown Adipose Tissue*

Recent work indicates that multiple molecular forms of Gs alpha can be produced by alternative splicing of a primary Gs alpha transcript. This study examined the pattern of expression of messenger RNAs (mRNAs) encoding the large (Gs alpha-L) and small (Gs alpha-S) molecular forms of Gs alpha in several rat tissues, including brown adipose tissue. In all tissues examined, Gs alpha-L mRNA was most abundant. This was most pronounced for brain tissue (95% of total), and least for brown adipose tissue (57%). The pattern of Gs alpha isoform expression of a given tissue was strongly correlated with the Gs alpha mRNA splicing pattern in that tissue. Cold exposure and surgical denervation are treatments that have been shown to, respectively, increase or decrease total Gs alpha mRNA in rat brown adipose tissue. Despite producing up to 4-fold differences in total Gs alpha mRNA, these treatments did not affect the proportions of Gs alpha mRNA splice variants in brown fat. In contrast, perinatal stimulation of brown fat was associated with changes in the splicing pattern of Gs alpha mRNA, and these changes were reflected in Gs alpha protein expression. Specifically, during the perinatal period Gs alpha-L mRNA increased significantly but Gs alpha-L protein levels did not change, whereas Gs alpha-S mRNA did not change and Gs alpha-S protein levels significantly declined. These data provide further evidence that the increased Gs alpha mRNA levels that occur in interscapular brown adipose tissue during periods of stimulation serve to maintain membrane levels of Gs alpha protein.

Two Distinct Tumor-Derived, Parathyroid Hormone-Like Peptides Result from Alternative Ribonucleic Acid Splicing

A novel PTH-like peptide has recently been isolated and cloned from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The deduced product of the initial clones to be reported is a 177 amino-acid protein, consisting of a 36 amino-acid precursor sequence followed by a 141 amino-acid mature peptide. Southern analysis of genomic DNA is compatible with a single-copy gene, but Northern analysis of mRNAs from both tumors and normal tissues consistently reveals multiple hybridizing transcripts, suggesting the possibility of alternative RNA splicing. We provide here direct evidence of alternative RNA splicing by the identification of a second cDNA clone in a human renal carcinoma cDNA library. As compared to the initial clone, this cDNA contains a foreshortened 5'-untranslated region but is otherwise identical until the distal portion of the coding region, at which point it diverges completely to encode a 166 amino-acid mature peptide with 27 amino acids of unique C-terminal sequence. The relative lengths of the primary translation products encoded by these two cDNAs were confirmed by transcription and translation in vitro, and both products were shown to be processed by added microsomes. The unique 3'-ends of these two cDNAs, as well as that of a third cDNA isolated by another laboratory, were used to identify one or more hybridizing transcripts corresponding to each cDNA in mRNA from a human renal carcinoma as well as in mRNA from normal human keratinocytes. We conclude that alternative RNA splicing results in mRNAs which encode multiple PTH-like peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the rat insulin-like growth factor II transcriptional unit: heterogeneous transcripts are generated from two promoters by use of multiple polyadenylation sites and differential ribonucleic acid splicing.

The rat insulin-like growth factor II (rIGF-II) gene, which exists as a single copy in the genome, is expressed as a multitranscript family of mRNA molecules ranging in size from 4.6 to 1 kilobases. Part of this heterogeneity can be ascribed to the presence of two different promoters, each transcribing alternative 5'-noncoding regions which are spliced to common coding exons. In the present study we use a combination of DNA sequence analysis of the gene, mapping of the mRNA molecules by Northern analysis and ribonuclease protection experiments, and DNA sequence analysis of cDNA clones complementary to different regions of the genome to establish the structure of several rIGF-II mRNA species. These results indicate that RNA heterogeneity also arises from the use of different polyadenylation sites. In addition, a variant 2 kilobases RNA was observed that was colinear with the distal 1700 base pairs of the 3147 base pair long exon 3, and may arise by alternative RNA splicing. These posttranscriptional modifications of RNAs arising from the rIGF-II transcription unit may generate molecules with different functional potential.

Ribonucleic acid splicing in Neurospora Mitochondria: secondary structure of the 35S ribosomal precursor ribonucleic acid investigated by digestion with ribonuclease III and by electron microscopy.

In Neurospora, the gene encoding the mitochondrial large (25S) ribosomal ribonucleic acid (rRNA) contains an intervening sequence of approximately 2.3 kilobases (kb). We have identified two temperature-sensitive mutants (289-67 and 299-9) which are defective in a factor encoded by a nuclear gene but required for the splicing of 25S RNA. When grown at the nonpermissive temperature (37 degrees C), the mutants accumulate a novel 35S RNA (5.2-5.6 kb) which is related to the natural precursor of 25S RNA and which has been shown to be a collinear transcript of the 25S RNA gene including the intervening sequence. In the present work, the secondary structure of 35S RNA was investigated by digestion with ribonuclease III and by electron microscopy of the RNA spread under partially denaturing conditions. Ribonuclease III cleaves 35S RNA predominantly at a central site or sites near the 5'-intron-exon boundary and produces fragments which correspond roughly to half-molecules (2.5-3 kb). Electron microscopy of 35S RNA shows a relatively large, central hairpin (180 +/- 45 nucleotides), which presumably corresponds to the central ribonuclease III site, and few other secondary structure features. Both experimental approaches indicate that the large hairpin is not present in 35S RNA. From this finding and from the location of the hairpin near the 5'-intron-exon boundary in 35S RNA, we infer that its formation requires intron sequences. 35S RNA from the mutants can be isolated as a ribonucleoprotein particle associated with almost the full complement of large subunit ribosomal proteins. The 35S RNA in such particles can be cleaved by ribonuclease III at the central site(s), consistent with the idea that the central hairpin is accessible to RNA-processing enzymes in vivo.