Alternative splicing makes a major contribution to proteomic diversity in higher eukaryotes with ~70% of genes encoding two or more isoforms. In most cases, the molecular mechanisms responsible for splice site choice remain poorly understood. Here, we used a randomization-selection approach in vitro to identify sequence elements that could silence a proximal strong 5′ splice site located downstream of a weakened 5′ splice site. We recovered two exonic and four intronic motifs that effectively silenced the proximal 5′ splice site both in vitro and in vivo. Surprisingly, silencing was only observed in the presence of the competing upstream 5′ splice site. Biochemical evidence strongly suggests that the silencing motifs function by altering the U1 snRNP/5′ splice site complex in a manner that impairs commitment to specific splice site pairing. The data indicate that perturbations of non-rate limiting step(s) in splicing can lead to dramatic shifts in splice site choice.

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A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans ( Communicated by Lester 0. Krampitz, July 25, 1988 (received for review June 6, 1988 ABSTRACTThe mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt sequence are transcribed to yield a 109-nt nonpolyadenylylated RNA with the 22-nt leader sequence at its 5' end. We suggest that the 22-nt leader is acquired by 63-kDa antigen mRNAs through trans-splicing. These results indicate that trans-splicing is widespread in nematodes and argue for the functional significance of the 22-nt spliced leader exon in nematode mRNA metabolism.Evidence suggests that intermolecular (trans) splicing is used in a variety of organisms during the maturation of some mRNAs. This is particularly clear for trypanosomatid protozoans, where all mRNAs contain a common leader derived from a small nonpolyadenylylated miniexon transcript (for review, see ref. 1). A trans-splicing mechanism of leader addition is supported by the primary structure of the miniexon transcript and the existence of appropriate branched intermediates (2, 3). Recent observations indicate that transsplicing might also be used in the formation of mRNA for chloroplast ribosomal protein S12 (4) and in the maturation of certain actin mRNAs in Caenorhabditis elegans (5).In C. elegans, mRNAs derived from three of four actin genes contain a 22-nucleotide (nt) leader sequence that is not encoded within 15 kilobases (kb) of the actin genes. This leader sequence is found as the first 22 nt of an abundant 100-base RNA transcribed from within the 5S rRNA gene cluster (5). Several lines of evidence, including the demonstration of branched intermediates containing a portion of the 100-nt RNA, suggest that the 22-nt leader is acquired by trans-splicing (5, 16). In contrast to the situation in trypanosomes, only a subset of C. elegans mRNAs appear to contain the trans-spliced leader. Furthermore, because C. elegans actin genes contain multiple introns, trans-splicing apparently occurs in conjunction with conventional cis-splicing. As discussed by Krause and Hirsh (5) the use of trans-splicing in C. elegans raises the possibility that this mechanism could be widespread in eukaryotes and may be a regulatory mechanism in gene expression.We have recently described the isolation and characterization of cDNA and genomic clones encoding a 63-kDa protective antigen in the human parasitic nematode Brugia malayi, the causative agent of lymphatic filariasis (6, 7).Nuclease protection and primer-extension experime...

Pre-messenger-RNA maturation in nematodes and in several other lower eukaryotic phyla involves spliced leader (SL) addition trans-splicing. In this unusual RNA processing reaction, a short common 5' exon, the SL, is affixed to the 5'-most exon of multiple pre-mRNAs. The nematode SL is derived from a trans-splicing-specific approximately 100-nucleotide RNA (SL RNA) that bears striking similarities to the cis-spliceosomal U small nuclear RNAs U1, U2, U4 and U5 (refs 3, 4); for example, the SL RNA functions only if it is assembled into an Sm small nuclear ribonucleoprotein (snRNP). Here we have purified and characterized the SL RNP and show that it contains two proteins (relative molecular masses 175,000 and 30,000 (M(r) 175K and 30K)) in addition to core Sm proteins. Immunodepletion and reconstitution with recombinant proteins demonstrates that both proteins are essential for SL trans-splicing; however, neither protein is required either for conventional cis-splicing or for bimolecular (trans-) splicing of fragmented cis constructs. The M(r) 175K and 30K SL RNP proteins are the first factors identified that are involved uniquely in SL trans-splicing. Several lines of evidence indicate that the SL RNP proteins function by participating in a trans-splicing specific network of protein protein interactions analogous to the U1 snRNP SF1/BBP U2AF complex that comprises the cross-intron bridge in cis-splicing.

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a -110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.A subset of mRNAs in the nematode Caenorhabditis elegans contains 22-nucleotide spliced-leader (SL) sequences at the 5' ends (2, 9). Several lines of evidence, including the identification of appropriate branched intermediates, indicate that the SL is acquired through an intermolecular (trans) splicing reaction (1, 9). In this reaction, the SL is donated from the 5' end of an -100-base RNA (SL RNA) transcribed from within the 5S rRNA gene cluster (9). The SL RNA of C. elegans has many features in common with well-characterized small nuclear RNAs (snRNAs) known to be essential for normal cis splicing. The SL RNA contains a trimethylguanosine (m32-7G) cap and has a consensus binding site (RAUnGR) for Sm, an antigen associated with small nuclear ribonucleoproteins (4,5,14,15). This Sm binding site is functional, since SL RNA is immunoprecipitated by anti-Sm antisera from extracts of C. elegans or after incubation in HeLa cell nuclear extracts (5,14,15). Furthermore, the sequence elements presumed to mediate transcriptional initiation and termination of SL RNA resemble those of vertebrate snRNAs (5). These properties suggest that the SL RNA might have a dual function in the trans-splicing process in which the 5' donated exon is covalently linked to an snRNA-like sequence (5, 15).trans splicing in nematodes does not appear to be restricted to C. elegans. Some mRNAs in the filarial parasite Bruigia malayi contain a 22-base SL identical in sequence to the C. elegans SL (13). In B. malayi, as in C. elegans, the SL is acquired from an SL RNA transcribed from within the 5S rRNA gene cluster (13 To examine the organization of SL-related sequences in Ascaris spp., an oligonucleotide complementary to the 22-nucleotide SL was used to probe Southern blots of genomic DNA digested with a variety of restriction enzymes. Parallel blots were also probed with an oligonucleotide complementary to B. mnalavi SS rRNA, since in six of the seven nematodes so far examined, the SL sequence is encoded within the 5S rRNA locus (2, 13). In DNA digested with ScaI, ClaI, or HaeIII, both probes identified a reiterated 1-kilobase fragment (data not shown). This result suggested that the SL-related sequence was linked to the 5S rRNA sequence in Ascaris spp. To characterize the SL sequence in detail, an Ascaris genomic library (in X EMBL4) (3) was screened by hybridization with both the SL and SS oligonucleotides. Plaques which hybridized to both probes were selected and purified by three successive roun...

Cytochrome P450 46A1 encoded by CYP46A1 catalyzes cholesterol 24-hydroxylation and is a CNS-specific enzyme that controls cholesterol removal and turnover in the brain. Accumulating data suggest that increases in cytochrome P450 46A1 activity in mouse models of common neurodegenerative diseases affect various, apparently unlinked biological processes and pathways. Yet, the underlying reason for these multiple enzyme activity effects is currently unknown. Herein, we tested the hypothesis that cytochrome P450 46A1-mediated sterol flux alters physico-chemical properties of the plasma membranes and thereby membrane-dependent events. We used 9-month-old 5XFAD mice (an Alzheimer’s disease model) treated for 6 months with the anti-HIV drug efavirenz. These animals have previously been shown to have improved behavioural performance, increased cytochrome P450 46A1 activity in the brain, and increased sterol flux through the plasma membranes. We further examined 9-month-old Cyp46a1−/− mice, which have previously been observed to have cognitive deficits and decreased sterol flux through brain membranes. Synaptosomal fractions from the brain of efavirenz-treated 5XFAD mice had essentially unchanged cholesterol levels as compared to control 5XFAD mice. However with efavirenz treatment in these mice, there were changes in the membrane properties (increased cholesterol accessibility, ordering, osmotic resistance and thickness) as well as total glutamate content and ability to release glutamate in response to mild stimulation. Similarly, the cholesterol content in synaptosomal fractions from the brain of Cyp46a1−/− mice was essentially the same as in wild-type mice but knockout of Cyp46a1 was associated with changes in membrane properties and glutamate content and its exocytotic release. Changes in Cyp46a1−/− mice were in the opposite direction to those observed in efavirenz-treated versus control 5XFAD mice. Incubation of synaptosomal fractions with the inhibitors of glycogen synthase kinase 3, cyclin-dependent kinase 5, protein phosphatase 1/2 A, and protein phosphatase 2B revealed that increased sterol flux in efavirenz-treated versus control 5XFAD mice affected the ability of all four enzymes to modulate glutamate release. In contrast, in Cyp46a1−/− versus wild-type mice, decreased sterol flux altered the ability of only cyclin-dependent kinase 5 and protein phosphatase 2B to regulate the glutamate release. Collectively, our results support cytochrome P450 46A1-mediated sterol flux as an important contributor to the fundamental properties of the membranes, protein phosphorylation and synaptic transmission. Also, our data provide an explanation of how one enzyme, cytochrome P450 46A1, can affect multiple pathways and processes and serve as a common potential target for several neurodegenerative disorders.

The trans‐spliced leader RNA (SL RNA) of nematodes resembles U snRNAs both in cap structure and in the presence of a consensus Sm binding site. We show here that synthetic SL RNA, synthesized by in vitro transcription, is efficiently used as a spliced leader donor in trans‐splicing reactions catalyzed by a cell free extract prepared from developing embryos of the parasitic nematode, Ascaris lumbricoides. Efficient utilization of synthetic SL RNA requires a functional Sm binding site. Mutations within the Sm binding sequence that prevent immunoprecipitation by Sm antisera and prevent cap trimethylation abolish trans‐splicing. The effect on trans‐splicing is not due to undermethylation of the cap structure.

Neuropeptide Y (NPY) levels are increased in plasma and tumors of patients with pheochromocytoma. The present study was designed to evaluate plasma and tissue NPY levels simultaneously as well as to study its release and expression in patients with either adrenal or extraadrenal pheochromocytomas.Plasma NPY levels were higher (P < 0.01) in patients with adrenal tumors than in matched normal subjects and patients with extraadrenal tumors. NPY levels were also higher (P < 0.05) in adrenal than in extraadrenal tumors. Bioactive NPY(1-36) was the predominant form in plasma and tumors of patients with adrenal pheochromocytomas. In contrast, patients with extraadrenal pheochromocytomas had an abundance of NPY fragments. NPY mRNA was abundant in 11 of 13 adrenal tumors but in only 1 of 6 extraadrenal tumors. Moreover, NPY was coreleased with NE with manipulation of adrenal but not extraadrenal tumors.These findings indicate that increased NPY gene expression in adrenal pheochromocytomas accounts for the greater biosynthesis and storage of NPY in these tumors and that increased release of NPY results in elevated plasma NPY. Factors regulating NPY gene expression in pheochromocytoma and the role of NPY in the clinical manifestations of the disease remain to be elucidated. (J. Clin. Invest. 1995.