A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).

Measurement of precursor miRNA in exosomes from human ESC-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) derived from human embryonic stem cells (ESCs) have been shown to secrete exosomes that are cardioprotective against myocardial ischemia reperfusion injury in a mouse model. To elucidate this cardioprotective mechanism, we have characterized the protein, nucleic acid, and lipid composition of MSC exosomes. Here we describe the isolation and analysis of RNA in MSC exosome. We have previously reported that RNAs in MSC exosome are primarily small RNA molecules of <300 nt and they include many miRNAs. Many of these miRNAs are in the precursor form suggesting that pre-miRNAs, and not mature miRNAs are preferentially loaded into exosomes. The protocols described here include assays to ascertain the presence of pre-miRNAs, profiling of miRNA and pre-miRNA, and quantitative estimation of mature and pre-miRNA.

Antisense-mediated exon skipping to shift alternative splicing to treat cancer.

Manipulation of alternative splicing is a unique way of regulating gene expression. Here, a detailed protocol is presented for modification of HER2 pre-mRNA alternative splicing. A 20-mer splice switching oligonucleotide (SSO) targeting exon 15 in HER2 pre-mRNA induced skipping of exon 15, resulted in significant downregulation of full-length HER2 mRNA and protein expression in HER2-overexpressing breast cancer cell line SK-BR-3.

Approaches to study CFTR pre-mRNA splicing defects.

In cystic fibrosis, genomic variants can result in defective processing of the CFTR precursor mRNA. Due to the complexity of the splicing process, the evaluation of their pathological effect is an important aspect both in the diagnostic field and in the study of basic regulatory mechanism. Efficient and correct splicing of CFTR relies on a complex process that includes recognition within the nascent transcripts of a series of different splicing regulatory elements that frequently overlap with the coding sequences. Identification of these elements is essential to determine the pathological impact of splicing-affecting genomic variants. In this chapter, to evaluate the effect of CFTR DNA variations on the pre-mRNA splicing process, different tools based on hybrid minigenes will be described.

The group II intron ribonucleoprotein precursor is a large, loosely packed structure.

Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to be the ancestors of spliceosomal introns and retrotransposons that insert into DNA. Folding of group II intron RNA is often guided by an intron-encoded protein to form a catalytically active ribonucleoprotein (RNP) complex that plays a key role in the activity of the intron. To date, possible structural differences between the intron RNP in its precursor and spliced forms remain unexplored. In this work, we have trapped the native Lactococcus lactis group II intron RNP complex in its precursor form, by deleting the adenosine nucleophile that initiates splicing. Sedimentation velocity, size-exclusion chromatography and cryo-electron microscopy provide the first glimpse of the intron RNP precursor as a large, loosely packed structure. The dimensions contrast with those of compact spliced introns, implying that the RNP undergoes a dramatic conformational change to achieve the catalytically active state.

Gonadotropin-inhibitory hormone neurons interact directly with gonadotropin-releasing hormone-I and -II neurons in European starling brain.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic dodecapeptide (SIKPSAYLPLRF-NH(2)) that directly inhibits gonadotropin synthesis and release from quail pituitary. The action of GnIH is mediated by a novel G-protein coupled receptor. This gonadotropin-inhibitory system may be widespread in vertebrates, at least birds and mammals. In these higher vertebrates, histological evidence suggests contact of GnIH immunoreactive axon terminals with GnRH neurons, thus indicating direct regulation of GnRH neuronal activity by GnIH. In this study we investigated the interaction of GnIH and GnRH-I and -II neurons in European starling (Sturnus vulgaris) brain. Cloned starling GnIH precursor cDNA encoded three peptides that possess characteristic LPXRF-amide (X = L or Q) motifs at the C termini. Starling GnIH was further identified as SIKPFANLPLRF-NH(2) by mass spectrometry combined with immunoaffinity purification. GnIH neurons, identified by in situ hybridization and immunocytochemistry (ICC), were clustered in the hypothalamic paraventricular nucleus. GnIH immunoreactive fiber terminals were present in the external layer of the median eminence in addition to the preoptic area and midbrain, where GnRH-I and GnRH-II neuronal cell bodies exist, respectively. GnIH axon terminals on GnRH-I and -II neurons were shown by GnIH and GnRH double-label ICC. Furthermore, the expression of starling GnIH receptor mRNA was identified in both GnRH-I and GnRH-II neurons by in situ hybridization combined with GnRH ICC. The cellular localization of GnIH receptor has not previously been identified in any vertebrate brain. Thus, GnIH may regulate reproduction of vertebrates by directly modulating GnRH-I and GnRH-II neuronal activity, in addition to influencing the pituitary gland.

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors.

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.

Identification of sea lamprey GTHbeta-like cDNA and its evolutionary implications.

We have identified the first and perhaps only gonadotropin beta-like protein by cDNA cloning in sea lamprey, a member of the oldest lineage of vertebrates, the agnathans. Two pituitary gonadotropins (GTHs: follicle-stimulating hormone (FSH) and luteinizing hormone (LH)) have been identified in representative species of all classes of vertebrates except the agnathans. The present study was undertaken to identify GTH in sea lamprey, Petromyzon marinus, to gain a further understanding of the origin and evolution of reproductive pituitary hormones and their respective genes in vertebrates. Sea lamprey preGTHbeta-like cDNA was cloned from a plasmid cDNA library using an expressed sequence tag analysis. The preGTHbeta-like cDNA encoded 150 amino acids, in which the GTHbeta-like protein consisted of 134 amino acid residues. Sea lamprey GTHbeta-like protein contained 12 Cys residues and two N-glycosylation sites at homologous positions to those of FSHbeta and LHbeta. The region of the molecule that has been proposed to control receptor binding specificity (i.e., the region between the 10th and 12th Cys residues) suggests that the proposed heterodimer would be more like a FSH than a LH. Sea lamprey GTHbeta-like protein-producing cells were identified immunocytochemically in the ventral part of the proximal pars distalis of pituitary using antiserum prepared against a synthetic peptide of preGTHbeta-like protein (52-68). Intraperitoneal administration of sea lamprey GnRH-I and -III at 100 microg/g body weight (twice at a 24h interval) increased expression of GTHbeta-like protein in the pituitary of adult female sea lamprey during the final maturational period. Thus, these results are the first to demonstrate the presence of a single GTH-like system in lampreys. Because the sea lamprey GTHbeta-like protein is a clear out-group compared to those of the LH and FSH family based on phylogenic analysis, we propose that an ancestral glycoprotein hormone gave rise to only one GTH in lampreys and to the glycoprotein hormone family that gave rise to LH, FSH, and TSH during the early evolution of gnathostomes.

Isolation and characterization of polyadenylation complexes assembled in vitro.

We developed a two-step purification of mammalian polyadenylation complexes assembled in vitro. Biotinylated pre-mRNAs containing viral or immunoglobulin poly(A) sites were incubated with nuclear extracts prepared from mouse myeloma cells under conditions permissive for in vitro cleavage and polyadenylation and the mixture was fractionated by gel filtration; complexes containing biotinylated pre-mRNA and bound proteins were affinity purified on avidin-agarose resin. Western analysis of known components of the polyadenylation complex demonstrated copurification of polyadenylation factors with poly(A) site-containing RNA but not with control RNA substrates containing either no polyadenylation signals or a point mutation of the AAUAAA polyadenylation signal. Polyadenylation complexes that were assembled on exogenous RNA eluted from the Sephacryl column in fractions consistent with their size range extending from 2 to 4 x 10(6) Mr. Complexes endogenous to the extract were of approximately the same apparent size, but more heterogeneous in distribution. This method can be used to study polyadenylation/cleavage complexes that may form upon a number of different RNA sequences, an important step towards defining which factors might differentially associate with specific RNAs.

Resolution of the mammalian E complex and the ATP-dependent spliceosomal complexes on native agarose mini-gels.

A great deal of progress in elucidating the mechanisms of spliceosome assembly has been achieved by analyzing the A, B, and C spliceosomal complexes on native polyacrylamide gels. In contrast, progress in understanding the earliest spliceosomal complex E has been hampered because this complex dissociates on native gels and is difficult to detect by other methods. Here we report conditions for resolving the spliceosomal complex E using a native horizontal agarose mini-gel system. This system also provides a simple alternative to polyacrylamide gels for resolving the ATP-dependent spliceosomal complexes.

Association of nonribosomal nucleolar proteins in ribonucleoprotein complexes during interphase and mitosis.

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin-a major nucleolar RNA-binding protein-contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

Identification and tissue distribution of human cystathionine beta-synthase mRNA isoforms.

Cystathionine beta-synthase (CBS) catalyzes the irreversible, serine-dependent conversion of homocysteine to cystathionine via a transsulfuration pathway. CBS deficiency not only is the leading cause of homocystinuria, an inherited genetic disorder, but may contribute to cardiovascular disease as well. We isolated three new isoforms of human CBS mRNA from a human liver cDNA library. We designate these CBS mRNAs as CBS 3, CBS 4, and CBS 5, and the CBS mRNAs reported previously by Kraus et al. (1993) (Hum. Mol. Genet. 2, 1933-1938) and Kruger and Cox (1994) (Proc. Natl. Acad. Sci. USA 91, 6614-6618) as CBS 1 and CBS 2, respectively. Sequence analyses show that the only difference among the five CBS mRNAs is at the beginning of the 5'-untranslated region. Tissue distribution studies reveal that liver and pancreas have the highest amounts of CBS mRNAs. CBS mRNA is present in all regions of the brain tested. We also report the differential distribution of CBS mRNA isoforms in tissues, showing that pancreas contains all five CBS isoforms and the liver has four CBS mRNA isoforms, CBS 1-4. The kidney contains only CBS 1 and CBS 2. In human fetal tissues, CBS 2 is present in the liver and kidney. PCR-based quantitative analyses of CBS mRNA isoforms in human liver demonstrate that CBS 1 and CBS 2 are the major species, with CBS 2 being more abundant, while CBS 3-5 are the minor species. Furthermore, results from our human liver cDNA screening and primer extension experiments show that each of the five CBS transcripts begins with a different exon, suggesting that CBS gene transcription might be regulated by more than one promoter.

Identification of a (CUG)n triplet repeat RNA-binding protein and its expression in myotonic dystrophy.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease that is associated with a (CTG)n repeat expansion in the 3'-untranslated region of the myotonin protein kinase (Mt-PK) gene. This study reports the isolation and characterization of a (CUG)n triplet repeat pre-mRNA/mRNA binding protein that may play an important role in DM pathogenesis. Two HeLa cell proteins, CUG-BP1 and CUG-BP2, have been purified based upon their ability to bind specifically to (CUG)8 oligonucleotides in vitro. While CUG-BP1 is the major (CUG)8-binding activity in normal cells, nuclear CUG-BP2 binding activity increases in DM cells. Both CUG-BP1 and CUG-BP2 have been identified as isoforms of a novel heterogeneous nuclear ribonucleoprotein (hnRNP), hNab50. The CUG-BP/hNab50 protein is localized predominantly in the nucleus and is associated with polyadenylated RNAs in vivo. In vitro RNA-binding/photocrosslinking studies demonstrate that CUG-BP/hNab50 binds to RNAs containing the Mt-PK 3'-UTR. We propose that the (CUG)n repeat region in Mt-PK mRNA is a binding site for CUG-BP/hNab50 in vivo, and triplet repeat expansion leads to sequestration of this hnRNP on mutant Mt-PK transcripts.

A mammalian activity required for the second step of pre-messenger RNA splicing.

Splicing of precursors to messenger RNAs occurs via a two-step mechanism. In the first step, the 5'-exon is released concomitant with the production of a lariat intermediate, and in the second step, the exons are joined, releasing the intron in the form of a lariat product. Several gene products of the yeast Saccharomyces cerevisiae have been shown to be required exclusively for the second step. Although mammalian proteins have been implicated in the second step of splicing, none have been shown to act only at this step. We identify here the first mammalian activity shown to be exclusively required for the second step. The activity was shown to increase by 5-fold the rate for this splicing step, whereas it had no effect on the rate of the first step. The activity was not affected by treatment with micrococcal nuclease, whereas it is sensitive to heating to 55 degrees C, suggesting that it is not dependent on an RNA, but more likely is a protein. The second step activity was separated from other factors required for the first step and from PSF, a splicing factor thought to have a second step activity. The activity does not require ATP hydrolysis, suggesting that it acts at a late stage of the second step of splicing.

Modulation of 5' splice site choice in pre-messenger RNA by two distinct steps.

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). Here, we perturbed the U1 snRNP in HeLa cell nuclear extract by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for 5' splice site selection. Accordingly, we found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. Our results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection.

Distinct functions of SR proteins in recruitment of U1 small nuclear ribonucleoprotein to alternative 5' splice sites.

Alternative splicing of precursor messenger RNAs (pre-mRNAs) is an important mechanism for the regulation of gene expression. The members of the SR protein family of pre-mRNA splicing factors have distinct functions in promoting alternative splice site usage. Here we show that SR proteins are required for the first step of spliceosome assembly, interaction of the U1 small nuclear ribonucleoprotein complex (U1 snRNP) with the 5' splice site of the pre-mRNA. Further, we find that individual SR proteins have distinct abilities to promote interaction of U1 snRNP with alternative 5' splice junctions. These results suggest that SR proteins direct 5' splice site selection by regulation of U1 snRNP assembly onto the pre-mRNA.

Characterization of the secondary structure of an oligonucleotide corresponding to the autocleavage site of a precursor RNA from bacteriophage T4.

We studied the secondary structure of an RNA fragment (GUUUCGUACAAAC) (R1) having the sequence corresponding to the self-cleavage domain in a precursor RNA molecule from bacteriophage T4 infected Escherichia coli cells (p2Sp1 RNA). We synthesized an oligoribonucleotide (CAAACGUACAAAC) (R3) which contained the sequence (CGUACA) proposed for the p2Sp1 RNA self-cleavage site but did not form the hairpin loop structure. The self-cleavage ability of the single stranded RNA (R3) is significantly lower than that of R1. We have also designed a modified RNA fragment (R2), which contained a noncleavable RNA with 2'-O-methylcytidine or 2-O-methyluridine. R3 did not exhibit cleavage. To further investigate the structural requirements in the cleavage reaction, we synthesized mutant RNAs which contained different bases within consensus sequences and the cleavage sites were tested for self-cleavage. Guanosine and adenosine 3'-phosphates seemed not to be susceptible to transesterification at the cleavage site. The data from native gel electrophoresis, the CD spectra and the Tm suggested that the hairpin structure is necessary for the cleavage.

Serine/threonine phosphorylation regulates binding of C hnRNP proteins to pre-mRNA.

The C hnRNP proteins bind to nascent pre-mRNA and are thought to participate in an early step of the pre-mRNA splicing pathway. We report here that C hnRNP proteins are phosphorylated by a casein kinase II activity in a HeLa cell nuclear extract and that dephosphorylation of C hnRNP proteins is inhibited by the specific protein-serine/threonine-phosphatase 1/2A inhibitor okadaic acid. We further find that dephosphorylation of C hnRNP proteins is required for their binding to adenovirus and human beta-globin pre-mRNAs. These results indicate that the participation of C hnRNP proteins in pre-spliceosome assembly is coupled to a dynamic cycle of their phosphorylation and dephosphorylation.

An ATP-independent U2 small nuclear ribonucleoprotein particle/precursor mRNA complex requires both splice sites and the polypyrimidine tract.

A complex is formed upon incubation of a precursor mRNA (pre-mRNA) with HeLA cell nuclear extract in the absence of added ATP (-ATP complex). Pre-mRNAs with mutations in the 5' splice site, the 3' splice site, or the polypyrimidine tract did not form this complex. Once formed, the -ATP complex was stable to competition by excess pre-mRNA. The complex was shown to contain the U2 small nuclear ribonucleoprotein particle (snRNP) and was distinct from the previously described U2 snRNP/pre-mRNA complex, the prespliceosome. These complexes have different electrophoretic mobilities, ATP requirements, and sensitivities to mutations of the 5' splice site. Although U1 snRNP was not found in the -ATP complex, a requirement for the U1 snRNP was suggested by immunodepletion experiments. The possible implications for the study of spliceosome formation are discussed.

Detection of human papillomavirus type 16 E6/E7 transcripts in fixed paraffin-embedded cervical cancers by the polymerase chain reaction.

A simple, and sensitive method for the detection of human papillomavirus (HPV) 16 E6/E7 transcripts in RNA purified from formalin acetic alcohol (FAA)-fixed, paraffin embedded cervical cancer (CaCx) tissue is described. The entire procedure, including polymerase chain reaction (PCR) amplification of cDNA obtained from mRNA by reverse transcription, can be completed within a day. The results obtained compare favourably with those using total nucleic acids extracted from the same fixed material, and are consistent with results using RNA purified from corresponding fresh tumour tissue. Although developed for investigating the expression of HPV in tumours, this method should find general application in studies of gene expression and viral detection.