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The RNP domain is found in a number of proteins involved in processing and transport of mRNA precursors. The crystal structure of a complex between the U1A spliceosomal protein and its cognate RNA hairpin at 1.92 A resolution reveals the molecular basis of sequence-specific RNA recognition by the RNP domain.
Although it has been known since the late 1970s that intron-containing and intronless versions of otherwise identical genes can exhibit dramatically different expression profiles, the underlying molecular mechanisms have only lately come to light. This review summarizes recent progress in our understanding of how introns and the act of their removal by the spliceosome can influence and enhance almost every step of mRNA metabolism. A rudimentary understanding of these effects can prove invaluable to researchers interested in optimizing transgene expression in eukaryotic systems.
BACKGROUND: In the translation of the genetic code each aminoacyl-tRNA synthetase (aaRS) must recognize its own (cognate) tRNA and attach the corresponding amino acid to the acceptor end of tRNA, discriminating all the others. The(alphabeta)2 phenylalanyl-tRNA synthetase (PheRS) is one of the most complex enzymes in the aaRS family and is characterized by anomalous charging properties. Structurally, the enzyme belongs to class II aaRSs, as its catalytic domain is built around an antiparallel beta sheet, but functionally it resembles class I as it aminoacylates the 2'OH of the terminal ribose of tRNA (class II aaRSs aminoacylate the 3'OH). With the availability of the three-dimensional structure of the complex between multisubunit PheRS and tRNAPhe, a fuller picture of the specific tRNA-aaRS interactions is beginning to emerge. RESULTS: The crystal structure of Thermus thermophilus PheRS complexed with cognate tRNA has been solved at 3.28 A resolution. It reveals that one tRNAPhe molecule binds across all four PheRS subunits. The interactions of PheRS with tRNA stabilize the flexible N-terminal part of the alpha subunit, which appeared to form the enzyme's 11th domain, comprising a coiled-coil structure (helical arm) built up of two long antiparallel alpha helices. The helical arms are similar to those observed in SerRS and are in the same relative orientation with respect to the catalytic domain. Anticodon recognition upon tRNA binding is performed by the B8 domain, the structure of which is similar to that of the RNA-binding domain (RBD) of the small spliceosomal protein U1A. The Th. thermophilus PheRS approaches the anticodon loop from the minor groove side. CONCLUSIONS: The mode of interactions with tRNA explains the absolute necessity for the (alphabeta)2 architecture of PheRS. The interactions of tRNAPhe with PheRS and particularly with the coiled-coil domain of the alpha subunit result in conformational changes in TPsiC and D loops seen by comparison with uncomplexed yeast tRNAPhe. The tRNAPhe is a newly recognized type of RNA molecule specifically interacting with the RBD fold. In addition, a new type of anticodon-binding domain emerges in the aaRS family. The uniqueness of PheRS in charging 2'OH of tRNA is dictated by the size of its adenine-binding pocket and by the local conformation of the tRNA's CCA end.
Although structural, biochemical, and genetic studies have provided much insight into the determinants of specificity and affinity of proteins for RNA, little is currently known about the kinetics that underlie RNA-protein interactions. Protein-RNA complexes are dynamic, and the kinetics of binding and release could influence many processes, such as the ability of RNA-binding proteins to compete for binding sites, the sequential assembly of ribonucleoprotein complexes, and the ability of bound RNA to move between cellular compartments. Therefore, to attain a complete and biologically relevant understanding of RNA-protein interactions, complex formation must be studied not only in equilibrated reactions, but also as a dynamic process. BIACORE, a surface plasmon resonance-based biosensor technology, allows intermolecular interactions to be measured in real time, and can provide both equilibrium and kinetic information about complex formation. This technology is a powerful tool with which to study the dynamics of RNA-protein interactions. We have used BIACORE extensively to obtain detailed insight into the interaction between RNA and proteins carrying RNA recognition motif domains. Here we discuss the physical principles on which BIACORE is based, and the required instrumentation. We describe how to design well-controlled RNA-protein interaction experiments aimed at yielding high-quality data, and outline the steps required for data analysis. In addition, we present examples to illustrate how kinetic studies have provided us with unique insights into the interaction of the spliceosomal U1A protein and the neuronal HuD protein with their respective RNA targets.
Much attention is currently being devoted to questions of protein and RNA tertiary structures and to the quaternary arrangement of the individual macromolecules in ribonucleoprotein (RNP) particles. In this article we describe two complementary strategies that allow the identification of RNA-protein contact sites in assembled, nonlabeled RNP particles after UV crosslinking. The first combines immunoprecipitation of UV-irradiated RNP particles under mildly denaturing conditions followed by primer-extension analysis of the crosslinked (and thus coprecipitated) RNA. The second involves the purification of crosslinked peptide-oligonucleotide from RNP particles and the subsequent analysis of the crosslinked peptide and RNA by Edman degradation and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS), respectively. Although the first approach provides a rapid method for the exact identification of RNA-protein contact sites in purified nonlabeled RNP particles, the latter adds valuable information about potential RNA binding domains within proteins and, thus, about the arrangement of these proteins within the quaternary structures of complex RNP assemblies. Recently, we applied both these strategies successfully to native purified spliceosomal RNP. These methods may be generally applicable to the analysis of RNP complexes, especially as they avoid labeling and reconstitution, both of which risk introducing artifacts.
Box C/D small nucleolar RNAs (snoRNAs) direct site-specific methylation of ribose 2'-hydroxyls in ribosomal and spliceosomal RNAs. To identify snoRNA functional groups contributing to assembly of an active box C/D snoRNP in Xenopus oocytes, we developed an in vivo nucleotide analog interference mapping procedure. Deleterious substitutions consistent with requirements for binding the 15.5 kD protein clustered within the terminal box C/D motif only. In vitro analyses confirmed a single interaction site for recombinant 15.5 kD protein and identified the exocyclic amine of A89 in box D as essential for binding. Our results argue that the 15.5 kD protein interacts asymmetrically with the two sets of conserved box C/D elements and that its binding is primarily responsible for the stability of box C/D snoRNAs in vivo.
Two types of eukaryotic nuclear introns are known: the common U2-dependent class with /GU and AG/ terminal intron dinucleotides, and the rare U12-dependent class with /AU and AC/ termini. Here we show that the U12-dependent splicing system can splice introns with /GU and AG/ termini and that such introns occur naturally. Further, U2-dependent introns with /AU and AC/termini also occur naturally and are evolutionarily conserved. Thus, the sequence of the terminal dinucleotides does not determine which spliceosomal system removes an intron. Rather, the four classes of introns described here can be sorted into two mechanistic classes (U2- or U12-dependent) by inspection of the complete set of conserved splice site sequences.
P-element somatic inhibitor (PSI) is a KH domain-containing splicing factor highly expressed in Drosophila somatic tissues. Here we have identified a direct association of PSI with the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) particle in somatic nuclear extracts. This interaction is mediated by highly conserved residues within the PSI C-terminal AB motif and the U1 snRNP-specific 70K protein. Through the AB motif, PSI modulates U1 snRNP binding on the P-element third intron (IVS3) 5' splice site and its upstream exonic regulatory element. Ectopic expression experiments in the Drosophila female germline demonstrate that the AB motif also contributes to IVS3 splicing inhibition in vivo. These data show that the processing of specific target transcripts, such as the P-element mRNA, is regulated by a functional PSI-U1 snRNP interaction in Drosophila.
The m(7)G caps of most spliceosomal snRNAs and certain snoRNAs are converted posttranscriptionally to 2,2,7-trimethylguanosine (m(3)G) cap structures. Here, we show that yeast Tgs1p, an evolutionarily conserved protein carrying a signature of S-AdoMet methyltransferase, is essential for hypermethylation of the m(7)G caps of both snRNAs and snoRNAs. Deletion of the yeast TGS1 gene abolishes the conversion of the m(7)G to m(3)G caps and produces a cold-sensitive splicing defect that correlates with the retention of U1 snRNA in the nucleolus. Consistently, Tgs1p is also localized in the nucleolus. Our results suggest a trafficking pathway in which yeast snRNAs and snoRNAs cycle through the nucleolus to undergo m(7)G cap hypermethylation.
The essential splicing factors SF1 and U2AF play an important role in the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. The structure of the C-terminal RRM (RRM3) of human U2AF(65) complexed to an N-terminal peptide of SF1 reveals an extended negatively charged helix A and an additional helix C. Helix C shields the potential RNA binding surface. SF1 binds to the opposite, helical face of RRM3. It inserts a conserved tryptophan into a hydrophobic pocket between helices A and B in a way that strikingly resembles part of the molecular interface in the U2AF heterodimer. This molecular recognition establishes a paradigm for protein binding by a subfamily of noncanonical RRMs.
Survival motor neuron (SMN) interacting protein 1 (SIP1) interacts with SMN protein and plays a crucial role in the biogenesis of spliceosomes. We have identified three novel splicing variants of the SIP1 (SIP1-beta, -gamma and -delta), in addition to the full-length SIP1-alpha. SIP1-alpha as found to be ubiquitously expressed at high levels in the various normal tissues examined. In contrast, SIP1-beta and -gamma were expressed at very low levels in these tissues. In muscle specimens from patients with spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), the expression of SIP1-alpha was dramatically decreased compared to that observed in the normal tissues. In addition, the expression of SIP1-beta was significantly increased in tissues derived from patients with either disease. These findings suggest that an aberrant alternative splicing event in SIP1 occurs tissues derived from patients with the motor neuron diseases, and contributes to the pathological process of SMA and ALS.
Most plant mRNAs are synthesized as precursors containing one or more intervening sequences (introns) that are removed during the process of splicing. The basic mechanism of spliceosome assembly and intron excision is similar in all eukaryotes. However, the recognition of introns in plants has some unique features, which distinguishes it from the reactions in vertebrates and yeast. Recent progress has occurred in characterizing the splicing signals in plant pre-mRNAs, in identifying the mutants affected in splicing and in discovering new examples of alternatively spliced mRNAs. In combination with information provided by the Arabidopsis genome-sequencing project, these studies are contributing to a better understanding of the splicing process and its role in the regulation of gene expression in plants.
In the past few years, our knowledge about small non-mRNAs (snmRNAs) has grown exponentially. Approaches including computational and experimental RNomics have led to a plethora of novel snmRNAs, especially small nucleolar RNAs (snoRNAs). Members of this RNA class guide modification of ribosomal and spliceosomal RNAs. Novel targets for snoRNAs were identified such as tRNAs and potentially mRNAs, and several snoRNAs were shown to be tissue-specifically expressed. In addition, previously unknown classes of snmRNAs have been discovered. MicroRNAs and small interfering RNAs of about 21-23 nt, were shown to regulate gene expression by binding to mRNAs via antisense elements. Regulation of gene expression is exerted by degradation of mRNAs or translational regulation. snmRNAs play a variety of roles during regulation of gene expression. Moreover, the function of some snmRNAs known for decades, has been finally elucidated. Many other RNAs were identified by RNomics studies lacking known sequence and structure motifs. Future challenges in the field of RNomics include identification of the novel snmRNA's biological roles in the cell.
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are spliceosomal macromolecular assemblages and thus actively participate in pre-mRNA metabolism. They are composed of evolutionarily conserved and tandemly repeated motifs, where both RNA-binding and protein-protein recognition occur to achieve cellular activities. By yet unknown mechanisms, these ribonucleoprotein (RNP) particles are targeted by autoantibodies and hence play significant role in a variety of human systemic autoimmune diseases. This feature makes them important prognostic markers in terms of molecular epidemiology and pathogenesis of autoimmunity. Since RNP domain is one of the most conserved and widespread scaffolds, evolutionary analyses of these RNA-binding domains can provide further clues on disease-specific epitope formation. The study presented herein represents a sequence comparison of RNA-recognition regions of recently cloned and characterized human hnRNP A3 with those of other relevant hnRNP A/B-type proteins. Their implications in human autoimmunity are particularly emphasized.
The rolA gene of Agrobacterium rhizogenes contains in its untranslated leader region a spliceosomal intron, which is spliced in Arabidopsis and in Nicotiana tabacum. Expression under the control of the 35S promoter from cauliflower mosaic virus of a rolA gene derivative defective in splicing still causes alterations of growth in transgenic tobacco plants. Splicing of rolA mRNA is required for efficient expression of the rolA phenotype in vivo. Moreover, splicing is required for efficient in vitro translation of the rolA mRNA. In contrast, expression of a 35S-rolA gene derivative with the ATG initiation codon replaced by ATA does not cause any phenotypical alteration. Mutations leading to amino acid substitutions at positions 37 and 40 of the rolA coding region were isolated as null mutants in Arabidopsis plants transgenic for the rolA gene. However, when expressed in tobacco under the control of the 35S promoter, they cause a rolA phenotype reduced in the expressivity of its traits. The molecular characterization of rolA mutants might be useful for understanding the biochemical function of the rolA protein.
The phylogenetic relationships of Tholurna dissimilis were investigated in relation to a phylogeny of twenty-three species in Caliciaceae and eighteen species from Physciaceae. ITS and LSU regions of the nuclear ribosomal DNA were used for the reconstruction of phylogenies by maximum parsimony methods. Calicium adaequatum was shown to be the closest relative of and possibly congeneric with Tholurna. Calicium is thus not monophyletic unless Tholurna is included. Calicium in the molecular phylogeny contains several distinct clades, which to some extent can be characterized morphologically. Cyphelium in a traditional sense is probably not monophyletic. Cyphelium s. str. has immersed apothecia, large smooth spores and a very thin excipulum throughout. C. inquinans and C. karelicum, which form a distinct and highly supported clade, may be accommodated in Acolium, possibly along with other Cyphelium and Calicium species. The phylogenies presented here do not support the recognition of neither Physciaceae nor Caliciaceae in a narrow sense, but they also do not exclude this. Numerous spliceosomal and unclassified insertions were found in the LSU sequences. They to some extent offered phylogenetic information both with respect to location and by their sequence similarities.
Nuclear bodies occurring during the 2-cell stage of bovine embryos (obtained either by in vitro fertilisation of in vitro matured ovarian oocytes, or collection after fertilisation and cleavage in vivo) were studied using ultrastructural cytochemistry and immunocytochemistry to determine whether their occurrence may be linked with the onset of embryonic transcription. In addition, the species-specific ultrastructural features of the interchromatin structures of the 2-cell bovine embryo were displayed. Three different types of nuclear bodies were distinguished: (i) nucleolus precursor bodies (NPBs), (ii) loose bodies (LBs) and (iii) dense bodies (DBs). In order to determine their possible functional significance, we considered parallels between these three nuclear entities and interchromatin compartments reported in other cells. As detected by their preferential ribonucleoprotein staining, all types of nuclear bodies contained ribonucleoproteins. In contrast to the other types of nuclear bodies studied, NPBs contained argyrophilic proteins but in no case they did show morphological features of functional nucleoli. Both compact and vacuolated forms of NPBs were seen in both in vivo and in vitro embryos, sometimes simultaneously in the same nucleus. LBs and DBs reacted with antibodies to Sm antigen, indicating the presence of a group of nucleoplasmic, non-nucleolar small nuclear ribonucleoproteins (snRNPs). The immunoreactivity for Sm antigen was more intense and homogeneous in DBs than in LBs. DBs were seen in both categories of embryo. A possible kinship of DBs with the sphere organelle known from oocytes of different animal species or the prominent spherical inclusions of the early mouse embryo nuclei is suggested. The last type of intranuclear body, the LBs, showed a composite structure. Their granular component, occurring in clusters and displaying immunoreactivity for Sm antigen, was similar to interchromatin granules and was therefore named IG-like granules. Another component forming the LBs showed a much finer structure and a lower immunoreactivity with anti-Sm antibodies. We suggest that this amorphous component may be related to the IG-associated zone. All three types of intranuclear bodies were often seen close together, suggesting their possible mutual functional relationship. From these and other observations we conclude that the intranuclear bodies in 2-cell bovine embryos correspond, with the exception of the NPB, to similar structures/compartments supposed to accumulate inactive spliceosomal components in certain phases of somatic cell nucleus functions. Accordingly, the occurrence of such nuclear bodies does not represent cytological evidence for RNA synthesis. In contrast to this, an important morphological feature revealing the status of the bovine 2-cell embryo is the vacuolisation of the NPB.
Base substitutions in U2/U6 helix I, a conserved base-pairing interaction between the U6 and U2 snRNAs, have previously been found to specifically block the second catalytic step of nuclear pre-mRNA splicing. To further assess the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3' splice site selection using a derivative of the yeast actin pre-mRNA. In these derivatives, the spacing between the branch site adenosine and 3' splice site has been reduced from 43 to 12 nt and this results in enhanced splicing of mutants in the conserved 3' terminal intron residue. In this context, mutation of the conserved 3' intron terminal G to a C also results in the partial activation of a nearby cryptic 3' splice site with U as the 3' terminal intron nucleotide. Using this highly sensitive mutant substrate, we have identified a mutation in the U6 snRNA (U57A) that significantly increases the selection of the cryptic 3' splice site over the normal 3' splice site and augments its utilization relative to that observed with the wild-type U2 or U6 snRNAs. In a previous study, we found that the same U6 mutation suppressed the effects of an A-to-G branch site mutation in an allele-specific fashion. The ability of U6-U57 mutants to influence the fidelity of both branch site and 3' splice site recognition suggests that this nucleotide may participate in the formation of the active site(s) of the spliceosome.