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Expression of tissue-restricted antigens (TRAs) within the thymus is critical for the establishment of self-tolerance; however, exact mechanisms regulating the expression of TRAs has proven more complex than previously appreciated. In this issue of theJCI, Muro et al. identify a central role for protein arginine methyltransferase 5 (PRMT5) in posttranscriptional regulation of TRAs and thereby central tolerance. Using conditional KO mice, the authors showed that thymic deficiency of PrmtS predisposed mice to developing autoimmune diseases while enhancing antitumor efficacy. These studies provide insight into the role of PRMT5 in shaping the T cell repertoire with implications for the development of therapies., Protein methyltransferase 5 Posttranslational modifications via arginine (R) methylation are catalyzed by a group of enzymes called protein arginine methyltransferases (PRMTs) and play an important role in gene transcription, cell-cycle [...]

Immunological self-tolerance is established in the thymus by the expression of virtually all self-antigens, including tissue-restricted antigens (TRAs) and cell-type-restricted antigens (CRAs). Despite a wealth of knowledge about the transcriptional regulation of TRA genes, posttranscriptional regulation remains poorly understood. Here, we show that protein arginine methylation plays an essential role in central immune tolerance by maximizing the self-antigen repertoire in medullary thymic epithelial cells (mTECs). Protein arginine methyltransferase-5 (Prmt5) was required for pre-mRNA splicing of certain key genes in tolerance induction, including Aire as well as various genes encoding TRAs. Mice lacking Prmt5 specifically in thymic epithelial cells exhibited an altered thymic T cell selection, leading to the breakdown of immune tolerance accompanied by both autoimmune responses and enhanced antitumor immunity. Thus, arginine methylation and transcript splicing are essential for establishing immune tolerance and may serve as a therapeutic target in autoimmune diseases as well as cancer immunotherapy., Introduction The thymus is the primary lymphoid organ that provides specialized microenvironments for generating the T cell repertoire (1). In 2 anatomically discrete regions within the thymus, the cortex and [...]

Cannabis saliva L. is widely used for fiber, medicinal, and other purposes, and many cultivars exist, yielding varying proportions of cannabinoids and terpenes. There is considerable interest in characterizing genomes and transcriptomes of C. sativa. Alternative splicing (AS) is a fundamental aspect of gene expression that results in multiple types of mRNAs produced by differential splicing. Transcriptome-wide identification of AS events in drug-type cultivars of C. sativa has not been reported. Here, we identified AS events using a transcriptome dataset derived from five drug-type cultivars with divergent chemotypes. Intron retention is the most common event type, followed by alternative acceptor, alternative donor, and skipped exons. We also sought to assess conservation of AS events among cultivars. We found 547 events (5%) unique to a single cultivar, 2661 (25%) shared by 2-4 cultivars, and 7569 (70%) common to all 5 cultivars. Genes with AS events in each set were analyzed for gene ontology enrichment, showing that genes with AS unique to a single cultivar are enriched for molecular functions related to interactions with ATP and processes involving transport within cells and across membranes. These results provide insights into the conservation and variation of AS events in multiple cultivars of C. sativa. Key words: Cannabis sativa L., alternative splicing, trichomes, transcriptome, 1 Introduction Alternative splicing is a process in which more than one splice site is recognized and used to cleave pre-mRNAs during transcription, creating two or more types of mature [...]

This study identifies MBNL proteins as negative regulators of alternative splicing events that are differentially regulated between ES cells and other cell types; several lines of evidence show that these proteins repress an ES cell alternative splicing program and the reprogramming of somatic cells to induced pluripotent stem cells. MBNL proteins and pluripotency Ben Blencowe and colleagues identify the muscleblind-like RNA binding proteins MBNL1 and MBNL2 as negative regulators of alternative splicing events that are differentially regulated between embryonic stem cells and other cell types. Several lines of evidence show that they are involved in the regulation of embryonic-stem-cell-like alternative splicing patterns. The authors also identify a regulatory role during the reprogramming of fibroblasts to induced pluripotent stem (iPS) cells. Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators.sup.1,2,3. Alternative splicing represents a widely acting mode of gene regulation.sup.4,5,6,7,8, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency.sup.9. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming., Author(s): Hong Han [sup.1] [sup.2] , Manuel Irimia [sup.1] , P. Joel Ross [sup.3] , Hoon-Ki Sung [sup.4] , Babak Alipanahi [sup.5] , Laurent David [sup.6] , Azadeh Golipour [sup.2] [...]

Single-cell RNA sequencing is used to investigate the transcriptional response of 18 mouse bone-marrow-derived dendritic cells after lipopolysaccharide stimulation; many highly expressed genes, such as key immune genes and cytokines, show bimodal variation in both transcript abundance and splicing patterns. This variation reflects differences in both cell state and usage of an interferon-driven pathway involving Stat2 and Irf7. Heterogeneity in immune cells Gene expression profiles are typically derived at cell-population level, yet there is growing evidence to suggest that seemingly identical individual cells can differ considerably in their gene expression. This paper describes the use of single-cell RNA sequencing (RNA-Seq) to analyse the transcriptional response of 18 mouse bone-marrow-derived dendritic cells after lipopolysaccharide stimulation. The authors find that even genes that are highly expressed at the population level -- such as key immune genes and cytokines -- are often bimodally expressed. They may be very highly expressed in one cell, and expressed hardly at all in another. This variation reflects differences in both cell state and usage of an interferon-driven pathway involving Stat2 and Irf7. The SMART-Seq technology used here could have wide application in the study of regulatory circuits at the single-cell level. Recent molecular studies have shown that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels and phenotypic output.sup.1,2,3,4,5, with important functional consequences.sup.4,5. Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs.sup.1,2 or proteins.sup.5,6 simultaneously, because genomic profiling methods.sup.3 could not be applied to single cells until very recently.sup.7,8,9,10. Here we use single-cell RNA sequencing to investigate heterogeneity in the response of mouse bone-marrow-derived dendritic cells (BMDCs) to lipopolysaccharide. We find extensive, and previously unobserved, bimodal variation in messenger RNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit, involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits., Author(s): Alex K. Shalek [sup.1] , Rahul Satija [sup.2] , Xian Adiconis [sup.2] , Rona S. Gertner [sup.1] , Jellert T. Gaublomme [sup.1] , Raktima Raychowdhury [sup.2] , Schraga Schwartz [...]

Alternative messenger RNA splicing is the main reason that vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription, and is greatly affected by the rate of transcript elongation (1-3). As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle; this is the functional form of the nascent pre-mRNA and determines the fate of the mature transcript (4). However, factors that connect the transcribing polymerase with the mRNP particle and help to integrate transcript elongation with mRNA splicing remain unclear. Here we characterize the human interactome of chromatin-associated mRNP particles. This led us to identify deleted in breast cancer 1 (DBC1) and ZNF326 (which we call ZNF-protein interacting with nuclear mRNPs and DBC1 (ZIRD)) as subunits of a novel protein complex--named DBIRD--that binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in (A + T)-rich DNA, and is present at the affected exons. RNA-interference-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. Together, these data indicate that the DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with the regulation of alternative splicing., The composition of mRNP particles has been the subject of a number of studies, using a variety of approaches (for example, ref. 5). There are likely to be different types [...]

The tra2[beta] gene encoding an alternative splicing regulator, transformer 2-[beta] (Tra2[beta]), generates five alternative splice variant transcripts (tra2[beta]1-5). Functionally active, full-length Tra2[beta] is encoded by tra2[beta]1 isoform. Expression and physiological significance of the other isoforms, particularly tra2[beta]4, are not fully understood. Rat gastric mucosa constitutively expressed tra2[beta]1 isoform and specifically generated tra2[beta]4 isoform that includes premature termination codon-containing exon 2, when exposed to restraint and water immersion stress. Treatment of a gastric cancer cell line (AGS) with arsenite (100 [micro]M) preferentially generated tra2[beta]4 isoform and caused translocation of Tra2[beta] from the nucleus to the cytoplasm in association with enhanced phosphorylation during the initial 4-6 h (acute phase). Following the acute phase, AGS cells continued upregulated tra2[beta]1 mRNA expression, and higher amounts of Tra2[beta] were reaccumulated in their nuclei. Treatment with small interference RNAs targeting up-frameshift-1 or transfection of a plasmid containing tra2[beta]1 cDNA did not induce tra2[beta]4 isoform expression and did not modify the arsenite-induced expression of this isoform, suggesting that neither the nonsense-mediated mRNA decay nor the autoregulatory control by excess amounts of Tra2[beta] participated in the tra2[beta]4 isoform generation. Knockdown of Tra2[beta] facilitated skipping of the central variable region of the CD44 gene and suppressed cell growth. In contrast, overexpression of Tra2[beta] stimulated combinatorial inclusion of multiple variable exons in the region and cell growth. The similar skipping and inclusion of the variable region were observed in arsenite-treated cells. Our results suggest that Tra2[beta] may regulate cellular oxidative response by changing alternative splicing of distinct genes including CD44. arsenite; splicing regulator; tra2[beta]4 isoform; premature termination codon; cell growth

From a large-scale screen using splicing microarrays and RT-PCR, we identified 63 alternative splicing (AS) events that are coordinated in 3 distinct temporal patterns during mouse heart development. More than half of these splicing transitions are evolutionarily conserved between mouse and chicken. Computational analysis of the introns flanking these splicing events identified enriched and conserved motifs including binding sites for CUGBP and ETR-3-like factors (CELF), muscleblind-like (MBNL) and Fox proteins. We show that CELF proteins are down-regulated >10-fold during heart development, and MBNL1 protein is concomitantly up-regulated nearly 4-fold. Using transgenic and knockout mice, we show that reproducing the embryonic expression patterns for CUGBP1 and MBNL1 in adult heart induces the embryonic splicing patterns for more than half of the developmentally regulated AS transitions. These findings indicate that CELF and MBNL proteins are determinative for a large subset of splicing transitions that occur during postnatal heart development. CUGBP and ETR-3-like factors | heart development | muscleblind-like | splicing microarray

Recently, it has been reported that there is a differential subcellular distribution of components of the minor U 12-dependent and major U2-dependent spliceosome, and further that the minor spliceosome functions in the cytoplasm. To study the subcellular localization of the snRNA components of both the major and minor spliceosomes, we performed in situ hybridizations with mouse tissues and human cells. In both cases, all spliceosomal snRNAs were nearly exclusively detected in the nucleus, and the minor U11 and U12 snRNAs were further shown to colocalize with U4 and U2, respectively, in human cells. Additionally, we examined the distribution of several spliceosomal snRNAs and proteins in nuclear and cytoplasmic fractions isolated from human cells. These studies revealed an identical subcellular distribution of components of both the U12- and U2-dependent spliceosomes. Thus, our data, combined with several earlier publications, establish that, like the major spliceosome, components of the U12-dependent spliceosome are localized predominantly in the nucleus. localization | pre-mRNA splicing | snRNA | U12-dependent spliceosome

Author(s): Caroline Heintz [1]; Thomas K. Doktor [2]; Anne Lanjuin [1]; Caroline C. Escoubas [1, 3]; Yue Zhang [1]; Heather J. Weir [1]; Sneha Dutta [1]; Carlos Giovanni Silva-Garca [1]; [...]

Pre-mRNA splicing not only removes introns and joins exons to generate spliced mRNA but also results in remodeling of the spliced messenger ribonucleoprotein, influencing various downstream events. This remodeling includes the loading of an exon-exon junction complex (EJC). It is unclear how the spliceosome recruits the EJC onto the mRNA and whether EJC formation or EJC components are required for pre-mRNA splicing. Here we immunodepleted the EJC core component eIF4A3 from HeLa cell nuclear extract and found that eIF4A3 is dispensable for pre-mRNA splicing in vitro. However, eIF4A3 is required for the splicing-dependent loading of the Y14/Magoh heterodimer onto mRNA, and this activity of human elF4A3 is also present in the Drosophila ortholog. Surprisingly, the loading of six other EJC components was not affected by eIF4A3 depletion, suggesting that their binding to mRNA involves different or redundant pathways. Finally, we found that the assembly of the EJC onto mRNA occurs at the late stages of the splicing reaction and requires the second-step splicing and mRNA-release factor HRH1/hPrp22. The EJC-dependent and -independent recruitment of RNA-binding proteins onto mRNA suggests a role for the EJC in messenger ribonucleoprotein remodeling involving interactions with other proteins already bound to the pre-mRNA, which has implications for nonsense-mediated mRNA decay and other mRNA transactions. messenger ribonucleoprotein remodeling nonsense-mediated mRNA decay | pre-mRNA splicing

Pre-mRNA splicing takes place in a large and highly dynamic complex known as the spliceosome. Here we report the optimization of a maltose-binding protein (MBP) affinity-purification method to isolate functional spliceosomes for electron microscopic analysis. Visualization of the spliceosome preparations revealed distinct 40-60 nm particles. Immunogold-conjugated antibodies to spliceosome components specifically label these particles, which are eliminated by treatment with either RNase or protease. Moreover, spliceosomes assembled on two different pre-mRNAs are indistinguishable. This first visualization of purified functional spliceosomes assembled in vitro reveals striking structural features, including one or more central cavities and multiple elongate lobes.

The author discusses the publications on fibrillin transcript. The topics of interest include the role of nonsense codon in the splice site choice and the effect of fibrillin transcript on exonic splicing enhancer.

Prp8 is the largest and most highly conserved protein in the spliceosome yet its mechanism of function is poorly understood. Our previous studies implicate Prp8 in control of spliceosome activation for the first catalytic step of splicing, because substitutions in five distinct regions (a-e) of Prp8 suppress a cold-sensitive block to activation caused by a mutation in U4 RNA. Catalytic activation of the spliceosome is thought to require unwinding of the U1 RNA/5' splice site and U4/U6 RNA helices by the Prp28 and Prp44/Brr2 DExD/H-box helicases, respectively. Here we show that mutations in regions a, d, and e of Prp8 exhibit allele-specific genetic interactions with mutations in Prp28, Prp44/Brr2, and U6 RNA, respectively. These results indicate that Prp8 coordinates multiple processes in spliceosome activation and enable an initial correlation of Prp8 structure and function. Furthermore, additional genetic interactions with U4-cs1 support a two-state model for this RNA conformational switch and implicate another splicing factor, Prp31, in Prp8-mediated spliceosome activation.

Research supports the view that the small nuclolar RNAs are the catalytic components of the spliceosome and bear resemblance to group II intron elements. Results reveal that the regions U2 and U6 are critical for catalysis in the spliceosome. The U6 gene contains intron and the RNA splicing reactions require magnesium.

Los1p, the Saccharomyces cerevisiae exportin-t homologue, binds tRNA and functions in pre-tRNA splicing and export of mature tRNA from the nucleus to the cytosol. Because LOS1 is unessential in yeast, other pathways for tRNA nuclear export must exist. We report that Cca1p, which adds nucleotides C, C, and A to the 3' end of tRNAs, is a multicopy suppressor of the defect in tRNA nuclear export caused by los1 null mutations. Mes1p, methionyl-tRNA synthetase, also suppresses the defect in nuclear export of tRN[A.sup.Met] in los1 cells. Thus, Cca1p and Mes1p seem to function in a Los1pindependent tRNA nuclear export pathway. Heterokaryon analysis indicates that Cca1p is a nucleus/cytosol-shuttling protein, providing the potential for Cca1p to function as an exporter or an adapter in this tRNA nuclear export pathway. In yeast, most mutations that affect tRNA nuclear export also cause defects in pre-tRNA splicing leading to tight coupling of the splicing and export processes. In contrast, we show that overexpressed Cca1p corrects the nuclear export, but not the pre-tRNA-splicing defects of los1::[Kan.sup.r] cells, thereby uncoupling pre-tRNA splicing and tRNA nuclear export.

The unfolded protein response pathway in endoplasmic reticulum is controlled by Hac1p transcription factor that is produced when intron at the 3'end of mRNA is removed. Data show that a base-pairing interaction between the intron and 5' region of mRNA blocks mRNA translation.

The yeast SUB2/yUAP56 gene roles in splicing are discussed in this research communication. The Schizosaccharomyces pombe ortholog of the UAP56 gene by heterologous complementation of a combined PRP40HA(sub.3)/nam 8(Delta) defect in budding yeast has been isolated. It was found that SUB2 participates in splicing in vivo and in vitro. U2snRNP likely has a role in events before prespiceosome formation.

Research shows that the RS domain of SF2/ASF protein is not required for in vitro splicing of constitutive and enhancer-dependent pre-mRNAs. Data indicate that specific substrates and strength of splicing signals determine the requirement of this domain.

Dual use of an acceptor/donor splice site has been found to govern alternative splicing of the interferon regulatory factor-3 (IRF-3) gene. The alternative spice isoform, IRF-3a, encoded by the IRF-3 gene is expressed in all tissues and cell lines tested. Ratios of IRF-3a to IRF3 mRNA are varied. Sequences downstream from the unusual splice junction can bind members of the ST protein family. This can affect splice site selection.

Research shows that RNA processing and degradation is regulated by RNase E enzyme. Data reveal that the amino teminal half of the enzyme is involved in the 5'-end -dependent RNA cleavage but not the carboxy-terminal half of RNase E.

In this review four small self-cleaving RNAs are described with reference to their structure and catalytic mechanisms. Data suggest that the mechanisms that RNA catalysts use are varied with the group I introns catalyzing a two-step reaction involving positioning of two distinct substrates in their respective active sites.

Evidence for a regulatory role of CUS2, a U2 suppressor protein, and a U2 snRNA structure in prespliceosome assembly has been found. ATP may only stimulate the rate of prespliceosome formation with CUS2 and U2 snRNA control. CUS2 brokers an interaction between PRP5 and the U2 snRNP that depends on the right U2 RNA structure. CUS2 seems to help couple PRP5 function to ATP-dependent steps in prespliceosome formation.

Results demonstrate that induction of unfolded protein response in mammalian cells involves proteolytic cleavage of transmembrane kinase/endoribonuclease Ire1 and that presenilin-1 controls Ire1 proteolysis.

Mechanisms of hair cell tuning are discussed with focus on features intrinsic to the hair cells that are active in tuning and mechanosensitivity. Special emphasis is placed on mechanical tuning the the transduction apparatus. Evidence that mammals employ electrical tuning over any frequency range has not been found. The active electromechanical output of the hair bundle may have intrinsic frequency limitations.

The spliceosome removes introns from pre-messenger RNAs by a mechanism that entails extensive remodeling of RNA structure. The most conspicuous rearrangement involves disruption of 24 base pairs between U4 and […]

Intron excision is an essential step in eukaryotic gene expression, but the molecular mechanisms by which the spliceosome accurately identifies splice sites in nuclear precursors to messenger RNAs (pre-mRNAs) are not well understood. A bimolecular assay for the second step of splicing has now revealed that exon ligation by the human spliceosome does not require covalent attachment of a 3' splice site to the branch site. Furthermore, accurate definition of the 3' splice site in this system is independent of either a covalently attached polypyrimidine tract or specific 3' exon sequences. Rather, in this system 3' splice site selection apparently occurs with a 5' → 3' directionality., Introns in nuclear pre-mRNAs are removed by the spliceosome, a 60S complex composed of the pre-mRNA, four small nuclear RNAs (snRNAs) (U1, U2, U4-U6, and U5), and numerous associated proteins [...]

A novel monoclonal antibody MC3 was utilized for the protein recognition of U2 snRNP auxiliary splicing factor (U2AF). The novel monoclonal antibody that was specific for U2AF produced a diffused stain throughout the nucleoplasm with additional concentration in nuclear speckles. The U2AF also colocalized with splicing small nuclear ribonucleoproteins and sarcoplasmic reticulum protein splicing factors in speckles that corresponded to the clusters of interchromatin granules within the nucleus.

The effects of alterations in the primary structure of three alpha1-isoforms on the functional properties of cardiac-type voltage-dependent Ca2+-channel (VDCC) were analyzed during primary transcript splicing. Alternative splicing of the COOH-terminal tail of L-type VDCC (L-VDCC) alpha1 subunits did not induce statistically significant differences in the behavior of the three alpha1 isoforms of L-VDCC. However, splice variants of the COOH terminus of the alpha1-subunit exhibited differential voltage-dependency to muscle Ca2+ antagonists.

Removal of a rare class of metazoan precursor messenger RNA introns with AU-AC at their termini is catalyzed by a spliceosome that contains U11, U12, and U5 small nuclear ribonucleoproteins. Two previously unidentified, low-abundance human small nuclear RNAs (snRNAs), U4atac and U6atac, were characterized as associated with the AT-AC spliceosome and necessary for AT-AC intron splicing. The excision of AT-AC introns therefore requires four snRNAs not found in the major spliceosome. With the use of psoralen crosslinking, a U6atac interaction with U12 was identified that is similar to a U6-U2 helix believed to contribute to the spliceosomal active center. The conservation of only limited U6atac sequences in the neighborhood of this interaction and the potential of U6atac to base pair with the 5' splice site consensus for AT-AC introns provide support for current models of the core of the spliceosome., The majority of eukaryotic precursor messenger RNA (pre-mRNA) introns contain sequences at their 5' and 3' splice sites that conform to the GU-AG consensus. Excision of these introns occurs in [...]

Nuclear messenger RNA splicing involves multiple interactions between the five spliceosomal small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5, and U6 and numerous spliceosomal proteins. Here it is shown that binding of a 2'-O-methyl-oligoribonucleotide complementary to U5 small nuclear RNA (snRNA) nucleotides 68 to 88 (BU5Ae) disrupts the initial U4/U5/U6 tri-snRNP complex, enhances the U2/U6 interaction, and induces a U1/U4/U5 snRNP complex. The U1/U4/U5 snRNP complex interacts specifically with an RNA oligonucleotide containing the 5' splice site sequence and may therefore represent a transitional stage in the displacement of U1 from the 5' splice site by U5 snRNP., Precursor mRNA (pre-mRNA) is splice to form mature mRNA by a dynamic complex, the spliceosome, composed of five snRNP particles (the U1, U2, U4, U5, an U6 snRNPs) and of [...]

The adult mouse testis contains two messenger RNA isoforms of the Sox17 gene that are formed by alternative splicing of the 5' untranslated domain and residues of an amino (NH2) -terminal. The Sox17 form encodes a Sry-related DNA-binding protein containing a high mobility group (HMG) box region near the NH2-terminus. The t-Sox17 form shows partial truncation of the HMG box region. Sox17 is expressed in spermatogonia and its expression decreases from early pachytene onwards. The t-Sox17 form is expressed from the pachytene stage onwards and is highly expressed in round spermatids.

Aminoglycoside inhibitors of translation have been shown previously to inhibit in vitro self-splicing by group I introns. Chemical probing of the phage T4-derived sunY intron shows that neomycin, streptomycin, and related antibiotics protected the N-7 position of G96, a universally conserved guanine in the binding site for the guanosine cofactor in the splicing reaction. The antibiotics also disrupted structural contacts that have been proposed to bring the 5' cleavage site of the intron into proximity to the catalytic core. In contrast, the strictly competitive inhibitors deoxyguanosine and arginine protected only the N-7 position of G96. Parallels between these results and previously observed protection of 16S ribosomal RNA by aminoglycosides raise the possibility that group I intron splicing and transfer RNA selection by ribosomes involve similar RNA structural motifs., Group I introns are a structurally related class of functional RNA elements that are capable of catalyzing their own excision and exon ligation in a well-characterized two-step process (1). Although [...]

The metabotropic glutamate receptors (mGluRs) comprise a second class of glutamate receptors which show variation by alternative splicing events. A splice variant of mGluR1a, termed mGluR1c, was isolated and characterized. The results showed that the the spliceevent replaced 312 C-terminal amino acids by a stretch of only 10 residues. Theparent and the variant receptors elicited different patterns of calcium releasein Xenopus oocytes. In addition, in situ hybridizations showed that mGluR1c is expressed at lower levels than other splice variants. These results show that the mGluR1 receptor can modify its function and distribution by alternative splicing.

A study was done on a fraction of the mRNA 5' cap-binding protein found in eukaryotic mRNAs. It was shown that the cap functions in translation, nucleocytoplasmic transport, splicing and stabilization of mRNA against 5' exonucleolytic degradation. Determination of cellular distribution of eIF-4E by means of indirect immunofluorescence shows that a fraction of the eIF-4E localizes in the nucleus which suggests its involvement in a nuclear function.

A highly sensitive and specific exon-trapping system was established using a specific plasmid vector wherein an exon is identified by its ability to remain as a mature RNA after splicing. The system was able to screen 300-kilobase-pair genomic DNA segments derived from human chromosomes 11q13. The chromosome contains genes responsible for human cancers and can be utilized to screen megabase genomic DNA segments for the presence of possible exons.

The N-methyl-D-aspartate (NMDA) receptors are channel forming glutamate receptors which have been proposed to be involved with long-term potentiation, synaptogenesis and developmental structuring. The cDNAs coding for the rat NMDAR1 receptors were isolated and characterized. The results showed that the receptors are variable, as a result of alternate splicing, with one variant, NR1b, differing from NR1a by a sequence insertion in the N-terminus and the presence of an alternate C-terminal domain. The structural differences result in differences in affinities for agonists and potentiation by spermine and protein kinase C.