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PubMedDate not supplied

Pb(II)-catalysed cleavage of the sugar-phosphate backbone of yeast tRNAPhe--implications for lead toxicity and self-splicing RNA.

Pb(II) is extremely efficient at depolymerizing RNA and studies on tRNAs have shown that site-specific cleavages in these molecules can be brought about by the action of Pb(II). We have observed, by difference Fourier analysis, sugar-phosphate strand scission between residues 17 and 18 in crystals of yeast tRNAPhe soaked in dilute Pb(II) solution at pH 7.4. We have also deduced the structure of the Pb(II)-tRNAPhe derivative at pH 5.0 where this cleavage reaction is considerably slower and report that, in this structure, the sugar-phosphate backbone remains intact. We have, therefore, a picture of the reactants (at pH 5.0) and products (at pH 7.4) of this cleavage reaction. From this crystallographic study, and associated biochemical work, we have formulated a possible mechanism for the cleavage reaction and also present here some general ideas on the action of metal ions on nucleic acids.

Humans↗
PubMed1996

The Pem homeobox gene. Androgen-dependent and -independent promoters and tissue-specific alternative RNA splicing.

The Pem gene encodes an atypical homeodomain protein, distantly related to Prd/Pax family members, that we demonstrate is regulated in a complex transcriptional and post-transcriptional manner. We show that the rat Pem genomic structure includes three 5'-untranslated (5'-UT) exons and four coding exons, three of which encode the homeodomain. Several alternatively spliced transcripts were identified, including one that skips an internal coding exon, enabling this mRNA to express a novel form of the Pem protein. Other alternatively spliced mRNAs were characterized that possess different 5'-UT regions, including a muscle-specific transcript. The different 5'-UT termini present in Pem transcripts conferred different levels of translatability in vitro. Two promoters containing multiple transcription initiation sites were identified: a distal promoter (Pd) in the first 5'-UT exon and a proximal promoter (Pp) located in the "intron" upstream of the first coding exon. The Pd was active in placenta, ovary, tumor cell lines, and to a lesser extent in skeletal muscle. In contrast, transcripts from the Pp were only detectable in testis and epididymis and were only expressed in epididymis in the presence of testosterone. To our knowledge no transcription factors have previously been identified that exhibit androgen-dependent expression in the epididymis.

Alternative Splicing↗
PubMed2025

The RNA splicing factor PRPF8 is required for left-right organiser cilia differentiation and determination of cardiac left-right asymmetry via regulation of Arl13b splicing.

Cilia function in the left-right organizer (LRO) is critical for determining internal organ asymmetry in vertebrates. To further understand the genetics of left-right asymmetry, we isolated a mouse mutant with laterality defects, l11Jus27, from a random mutagenesis screen. l11Jus27 mutants carry a missense mutation in the pre-mRNA processing factor, Prpf8. cephalophŏnus (cph) mutant zebrafish, carrying a protein truncating mutation in prpf8, phenocopy the laterality defects of l11Jus27 mutants. Prpf8 mutant mouse and fish embryos have increased expression of an alternative transcript encoding the cilium-associated protein, ARL13B, that lacks exon 9. In zebrafish, over-expression of the arl13b transcript lacking exon 9 perturbed cilium formation and caused laterality defects. The shorter ARL13B protein isoform lacked interactions with intraflagellar transport proteins. Our data suggest that PRPF8 plays a prominent role in LRO cilia by through the regulation of alternative splicing of ARL13B, thus uncovering a new mechanism for cilia-linked developmental defects.

ARL13B↗
PubMed1992

High Degree of NCAM Diversity Generated by Alternative RNA Splicing in Brain and Muscle.

The neural cell adhesion molecules (NCAMs) are cell surface glycoproteins involved in vertebrate cell contact formation. Several NCAM mRNA types are generated from a single primary transcript by alternative splicing and differential polyadenylation. In this presentation we analyse sequence heterogeneities within NCAM transcripts detected in the junctions of exons 7/8, 12/13 and 13/14. The highest degree of evolutionary conservation is observed in the 30-nucleotide insertion (pi) between exons 7 and 8, coding for an identical peptide sequence in the mouse, rat and chicken. The most complex splicing pattern is found between exons 12 and 13, called splice site a. Three alternative exons of 15, 48 and 42 nucleotides can be inserted in various combinations, which may also contain the additional trinucleotide AAG. In mouse muscle cell lines, differential 'extra exon' and AAG usage in splice site a creates up to 16 NCAM diversity forms, some (if not all) of which are also expressed in mouse brain. Additional microdiversity is generated by the insertion of an alternative AAG trinucleotide in exon junction 13/14. If all combinations of splicing patterns identified so far were to occur and to be translated, there could be up to 192 different NCAM proteins.

Journal Article↗
PubMed1995

Control of proton sensitivity of the NMDA receptor by RNA splicing and polyamines.

The function of the N-methyl-D-aspartate (NMDA)-preferring glutamate receptor can be regulated by extracellular pH, a process that may be important during ischemia in the brain or during seizures. Protons inhibit NMDA receptor function by 50 percent at pH 7.3 through interactions with the NR1 subunit, and both polyamines and NR1 exon 5 potentiate receptor function through relief of the tonic proton inhibition present at physiological pH. A single amino acid (lysine 211) was identified that mediates the effects of exon 5 in the rat brain. Electroneutral substitutions at this position restored pH sensitivity and, consequently, polyamine relief of tonic inhibition. This effect, together with the structural similarities between polyamines and the surface loop encoded by exon 5, suggest that exon 5 may act as a tethered pH-sensitive constitutive modulator of NMDA receptor function.

Alternative Splicing↗
PubMed1996

Alternative RNA splicing that determines agrin activity regulates binding to heparin and alpha-dystroglycan.

Agrin is a component of the extracellular matrix that regulates aspects of neuromuscular junction differentiation. Identification of agrin-binding proteins has lead to the suggestion that alpha-dystroglycan is a muscle cell surface proteoglycan that mediates agrin activity. To further test this hypothesis, we have compared the ability of differentially active agrin isoforms to interact with a model component of proteoglycans, heparin, as well as with the putative proteoglycan alpha-dystroglycan. We demonstrate that an alternately spliced exon (encoding the sequence lysine, serine, arginine, lysine: Y site) is necessary for agrin-heparin interactions. We also show that alternate splicing at another site (Z site) dramatically affects interaction of alpha-dystroglycan with agrin. We propose a model in which multiple distinct domains of agrin interact with both protein and sugar moieties of alpha-dystroglycan. The isoform-specific binding of agrin to alpha-dystroglycan is consistent with a functional role for this interaction during synaptogenesis.

Agrin↗
PubMed2000

Internally shortened menin protein as a consequence of alternative RNA splicing due to a germline deletion in the multiple endocrine neoplasia type 1 gene.

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominantly inherited cancer syndrome (OMIM 131100), with tumours in several endocrine glands. In 1997 the responsible tumour suppressor gene was identified and recently it was shown that menin, its encoded protein, represses JunD-activated gene expression. Although many MEN 1 patients have been investigated both clinically and genetically, no genotype-phenotype correlation has been found yet. The vast majority of MEN1 gene mutations involve point mutations. We describe a patient in whom a 26 base pair deletion in the MEN1 gene, comprising part of exon 3 and part of intron 3, causes activation of a cryptic donor splice site at the beginning of exon 3. This germline mutation results in an in frame deletion of 105 nucleotides in MEN1 gene mRNA, i.e. an internal deletion of 35 amino acids in the menin protein. Since the deleted region of menin has been implicated in binding to JunD, this may explain the tumourigenic effect of this mutation. The knowledge of this MEN1 gene germline defect, may be used for presymptomatic identification of MEN 1 disease gene-carriers among family-members of this proband. This enables early detection of tumour development, timely treatment and genetic counseling.

Adult↗
PubMed2005

Alternative RNA splicing and drug target identification.

Incorporating alternative splicing content into discovery stage DNA microarray expression profiling studies fundamentally increases the relevance of the information obtained. This is because alternative splicing content addresses different parameters to those of conventional microarray content, in that it detects changes that impact the structure and function of gene products, and not simply the quantity of transcription of a given gene. Furthermore, alternative splicing has evolved not as a random process, but as a regulator of protein-protein interactions by preferentially targeting interaction domains for removal, insertion or replacement. In addition, a considerable body of literature is now available linking alternative splicing to the inheritance and pathology of diseases. Alternative splicing affects nearly every gene, with an average of three expressed protein products per gene in humans. ExonHit Therapeutics SA has recently produced the first commercial line of nucleic acid microarrays with splice event resolution that allow detection of not only the reference form of the gene, but also of all the known alternative splice forms. These microarrays enable profiling of the molecular events that are missed with conventional microarrays, and thus permit the key issues of tissue distribution and functional and pharmacological effects of alternative splicing to be addressed earlier in discovery.

Alternative Splicing↗
PubMed1995

Molecular cloning and expression of multiple isoforms of human prostaglandin E receptor EP3 subtype generated by alternative messenger RNA splicing: multiple second messenger systems and tissue-specific distributions.

Five distinct cDNA clones encoding four different isoforms of human prostaglandin (PG) E receptor EP3 subtype were isolated from a human kidney cDNA library. Two cDNA clones differed only in their 3'-untranslated regions. The four isoforms, tentatively named EP3-I, EP3-II, EP3-III, and EP3-IV, which were generated by alternative mRNA splicing, had identical amino acid sequences except for their different carboxyl-terminal tails. Transfection experiments revealed that all the four isoforms show high binding affinities to PGE2, PGE1, and M&B28767, an EP3-specific agonist, whereas their downstream signaling pathways are divergent. M&B28767 increased cAMP concentrations in cells expressing EP3-II and EP3-IV, whereas it inhibited forskolin-induced cAMP accumulations in cells expressing all EP3 isoforms. M&B28767 also stimulated phosphoinositide turnover in cells expressing EP3-I and EP3-II. Northern blot analysis revealed that the EP3 gene is expressed in a wide variety of human tissues. The human EP3 mRNA was present most abundantly in the kidney, pancreas, and uterus. A substantial expression was also detected in the heart, liver, skeletal muscle, small intestine, colon, prostate, ovary, and testis. Furthermore, reverse transcription-polymerase chain reaction analysis demonstrated tissue-specific expressions of the five different EP3 mRNA species. The present study suggests the presence of the multiple systems of PGE2/EP3 isoforms and leads to the better understanding of its physiological and pathophysiological implications in humans.

Alternative Splicing↗
PubMed2000

Coupled in vitro synthesis and splicing of RNA polymerase II transcripts.

Compelling in vivo studies suggest a tight functional linkage between RNA polymerase II transcription and premessenger RNA splicing. At present, the specific interactions involved in this coupling are poorly understood and deserve investigation. To this end, we developed an in vitro system that permits study of coupled transcription and splicing. Transcripts generated by RNA polymerase II were accurately and efficiently spliced under reaction conditions that permitted both transcription and splicing to occur simultaneously. The splicing of RNA-polymerase-II-driven transcripts was accelerated relative to that of the same transcripts driven by T7 RNA polymerase. Moreover, the product of exon ligation was found associated with the DNA template in reactions driven by RNA polymerase II. These two findings indicate that transcription and splicing were coupled in the in vitro system driven by RNA polymerase II, and suggest that this system will be useful for the biochemical study of this coupling.

Base Sequence↗
PubMed2005

Spliceosome-mediated RNA trans-splicing.

RNA repair or reprogramming is a new avenue for human gene therapy. Unlike conventional gene therapy, in which exogenous cDNAs are introduced into cells, RNA repair approaches, which are based on spliceosome-mediated pre-mRNA trans-splicing, trans-splicing ribozymes, and tRNA-splicing endonuclease, allow the correction of endogenous RNA species. Recently published accounts that in vivo phenotypic correction of a variety of inherited diseases can be achieved by RNA repair are encouraging. Nevertheless, the science of RNA repair for treatment of human diseases is just beginning and faces several scientific and technical challenges that must be addressed and surmounted. In this review, we summarize recent advances in spliceosome-mediated pre-mRNA trans-splicing. We also provide an update on the progress of this emerging technology toward the development of molecular therapy and diagnosis for human diseases and discuss the outstanding issues and challenges confronting RNA therapeutics.

Animals↗