The conserved 3′ end domain of U6atac snRNA can direct U6 snRNA to the minor spliceosome

U6 and U6atac snRNAs play analogous critical roles in the major U2-dependent and minor U12-dependent spliceosomes, respectively. Previous results have shown that most of the functional cores of these two snRNAs are either highly similar in sequence or functionally interchangeable. Thus, a mechanism must exist to restrict each snRNA to its own spliceosome. Here we show that a chimeric U6 snRNA containing the unique and highly conserved 3′ end domain of U6atac snRNA is able to function in vivo in U12-dependent spliceosomal splicing. Function of this chimera required the coexpression of a modified U4atac snRNA; U4 snRNA could not substitute. Partial deletions of this element in vivo, as well as in vitro antisense experiments, showed that the 3′ end domain of U6atac snRNA is necessary to direct the U4atac/U6atac.U5 tri-snRNP to the forming U12-dependent spliceosome. In vitro experiments also uncovered a role for U4atac snRNA in this targeting.     

U6atac snRNA,the highly divergent counterpart of U6 snRNA,is the specific target that mediates inhibition of AT-AC splicing by the influenza virus NS1 protein.

The influenza virus NS1 protein inhibits the splicing of the major class of mammalian pre-mRNAs (GU-AG Introns) by binding to a specific stem-bulge in U6 snRNA, thereby blocking the formation of U4/U6 and U2/U6 complexes. The splicing of the minor class of AT-AC introns takes place on spliceosomes that do not contain U6 snRNA, but rather U6atac snRNA-a highly divergent U6 snRNA counterpart. Nonetheless, we demonstrate that the NS1 protein inhibits AT-AC splicing in vitro, and specifically binds to only U6atac snRNA among the five minor class snRNAs. Chemical modification/interference assays show that the NS1 protein binds to the stem-bulge near the 3'' end of U6atac snRNA, encompassing nt 82-95 and nt 105-114. Although the sequence of this stem-bulge differs significantly from the sequence of the stem-bulge to which the NS1 protein binds in U6 snRNA, RNA competition experiments Indicate that U6 and U6atac snRNAs likely share the same binding site on the NS1 protein. Previously, the region of U6atac snRNA containing this 3'' stem-bulge had not been implicated in any interactions of this snRNA with either U4atac or U12 snRNA. However, as assayed by psoralen crosslinking, we show that the NS1 protein inhibits the formation of U12/U6atac complexes, but not the formation of U4atac/U6atac complexes. We can conclude that the inhibition of AT-AC splicing results largely from the inhibition of formation of U12/U6atac complexes caused by the binding of the NS1 protein to the 3'' stem-bulge of U6atac snRNA.     

Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion

RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B″ proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the β-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B″ proteins. However, unlike U1A and U2B″, some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an α-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.     

Comprehensive in vivo RNA-binding site analyses reveal a role of Prp8 in spliceosomal assembly

Prp8 stands out among hundreds of splicing factors as a protein that is intimately involved in spliceosomal activation and the catalytic reaction. Here, we present the first comprehensive in vivo RNA footprints for Prp8 in budding yeast obtained using CLIP (cross-linking and immunoprecipitation)/CRAC (cross-linking and analyses of cDNAs) and next-generation DNA sequencing. These footprints encompass known direct Prp8-binding sites on U5, U6 snRNA and intron-containing pre-mRNAs identified using site-directed cross-linking with in vitro assembled small nuclear ribonucleoproteins (snRNPs) or spliceosome. Furthermore, our results revealed novel Prp8-binding sites on U1 and U2 snRNAs. We demonstrate that Prp8 directly cross-links with U2, U5 and U6 snRNAs and pre-mRNA in purified activated spliceosomes, placing Prp8 in position to bring the components of the active site together. In addition, disruption of the Prp8 and U1 snRNA interaction reduces tri-snRNP level in the spliceosome, suggesting a previously unknown role of Prp8 in spliceosomal assembly through its interaction with U1 snRNA.     

New tertiary constraints between the RNA components of active yeast spliceosomes: a photo-crosslinking study

Elucidation of the three-dimensional (3D) structures of the two sequential active sites in spliceosomes is essential for understanding the mechanism of premessenger RNA splicing. The mechanism is predicted to be catalyzed by the small nuclear RNA (snRNA) components of spliceosomes. To obtain new tertiary constraints between the RNA components, we produced and mapped crosslinks between U6 snRNA and the proximal RNAs of active yeast spliceosomes ("yeast" in this report is Saccharomyces cerevisiae). Thus, specific sites in U6, when substituted with a photoreactive 4-thiouridine or 5-iodouridine, produced spliceosome-dependent crosslinks to U2 snRNA, or in one case, to the pre-mRNA substrate. One set of U2-U6 crosslinks formed before the Prp2p-dependent step of spliceosome assembly, whereas another set formed during or after this step but before the first chemical step of splicing. This latter set of crosslinks formed across U2-U6 helix I. Importantly, this set provides new tertiary constraints for developing 3D models of fully assembled yeast spliceosomes, which are poised for the first chemical step of splicing.     

The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly

In the pre-mRNA processing machinery of eukaryotic cells, U6 snRNA is located at or near the active site for pre-mRNA splicing catalysis, and U6 is involved in catalyzing the first chemical step of splicing. We have further defined the roles of key features of yeast U6 snRNA in the splicing process. By assaying spliceosome assembly and splicing in yeast extracts, we found that mutations of yeast U6 nt 56 and 57 are similar to previously reported deletions of U2 nt 27 or 28, all within yeast U2-U6 helix Ia. These mutations lead to the accumulation of yeast A1 spliceosomes, which form just prior to the Prp2 ATPase step and the first chemical step of splicing. These results strongly suggest that, at a late stage of spliceosome assembly, the presence of U2-U6 helix Ia is important for promoting the first chemical step of splicing, presumably by bringing together the 5' splice site region of pre-mRNA, which is base paired to U6 snRNA, and the branchsite region of the intron, which is base paired to U2 snRNA, for activation of the first chemical step of splicing, as previously proposed by Madhani and Guthrie [Cell, 1992, 71: 803-817]. In the 3' intramolecular stem-loop of U6, mutation G81C causes an allele-specific accumulation of U6 snRNP. Base pairing of the U6 3' stem-loop in yeast spliceosomes does not extend as far as to include the U6 sequence of U2-U6 helix Ib, in contrast to the human U6 3' stem-loop structure.     

Mapping U2 snRNP--pre-mRNA interactions using biotinylated oligonucleotides made of 2''-OMe RNA.

Biotinylated 2'-OMe RNA oligonucleotides complementary to two separate regions of human U2 snRNA have been used as affinity probes to study U2 snRNP--pre-mRNA interactions. Both oligonucleotides bind specifically and allow highly selective removal of U2 snRNP from HeLa cell nuclear extracts. Pre-mRNA substrates can also be specifically affinity selected through oligonucleotides binding to U2 snRNP particles in splicing complexes. Stable binding of U2 snRNP to pre-mRNA is blocked by the pre-binding of an oligonucleotide to the branch site complementary region of U2 snRNA, but not by an oligonucleotide binding to the 5' terminus of U2. Both oligonucleotides affinity select the intron product, but not the intron intermediate, when added after spliceosome assembly has taken place. The effect of 2'-OMe RNA oligonucleotides on splicing complex formation has been used to demonstrate that complexes containing U2 snRNP and unspliced pre-mRNA are precursors to functional spliceosomes.     

Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis

We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.     

植物核小RNA研究进展

核小RNA (small nuclear RNA,snRA)是一类长度为60～300 nt的非编码RNA,是真核生物RNA剪接体的主要成分.snRNA在各种生物中具有较高的保守性和同源性,主要参与了mRNA和rRNA前体的加工过程.本文对当前植物中snRNA领域的研究成果进行简要概述,介绍了植物snRNA的分类、基因结...     

The RNA binding protein Cwc2 interacts directly with the U6 snRNA to link the nineteen complex to the spliceosome during pre-mRNA splicing

Intron removal during pre-messenger RNA (pre-mRNA) splicing involves arrangement of snRNAs into conformations that promote the two catalytic steps. The Prp19 complex [nineteen complex (NTC)] can specify U5 and U6 snRNA interactions with pre-mRNA during spliceosome activation. A candidate for linking the NTC to the snRNAs is the NTC protein Cwc2, which contains motifs known to bind RNA, a zinc finger and RNA recognition motif (RRM). In yeast cells mutation of either the zinc finger or RRM destabilize Cwc2 and are lethal. Yeast cells depleted of Cwc2 accumulate pre-mRNA and display reduced levels of U1, U4, U5 and U6 snRNAs. Cwc2 depletion also reduces U4/U6 snRNA complex levels, as found with depletion of other NTC proteins, but without increase in free U4. Purified Cwc2 displays general RNA binding properties and can bind both snRNAs and pre-mRNA in vitro. A Cwc2 RRM fragment alone can bind RNA but with reduced efficiency. Under splicing conditions Cwc2 can associate with U2, U5 and U6 snRNAs, but can only be crosslinked directly to the U6 snRNA. Cwc2 associates with U6 both before and after the first step of splicing. We propose that Cwc2 links the NTC to the spliceosome during pre-mRNA splicing through the U6 snRNA.     

Lsm proteins promote regeneration of pre-mRNA splicing activity

Lsm proteins are ubiquitous, multifunctional proteins that affect the processing of most RNAs in eukaryotic cells, but their function is unknown. A complex of seven Lsm proteins, Lsm2-8, associates with the U6 small nuclear RNA (snRNA) that is a component of spliceosome complexes in which pre-mRNA splicing occurs. Spliceosomes contain five snRNAs, U1, U2, U4, U5, and U6, that are packaged as ribonucleoprotein particles (snRNPs). U4 and U6 snRNAs contain extensive sequence complementarity and interact to form U4/U6 di-snRNPs. U4/U6 di-snRNPs associate with U5 snRNPs to form U4/U6.U5 tri-snRNPs prior to spliceosome assembly. Within spliceosomes, disruption of base-paired U4/U6 heterodimer allows U6 snRNA to form part of the catalytic center. Following completion of the splicing reaction, snRNPs must be recycled for subsequent rounds of splicing, although little is known about this process. Here we present evidence that regeneration of splicing activity in vitro is dependent on Lsm proteins. RNP reconstitution experiments with exogenous U6 RNA show that Lsm proteins promote the formation of U6-containing complexes and suggest that Lsm proteins have a chaperone-like function, supporting the assembly or remodeling of RNP complexes involved in splicing. Such a function could explain the involvement of Lsm proteins in a wide variety of RNA processing pathways.     

Mutation in the U2 snRNA influences exon interactions of U5 snRNA loop 1 during pre-mRNA splicing

The U2 and U6 snRNAs contribute to the catalysis of intron removal while U5 snRNA loop 1 holds the exons for ligation during pre-mRNA splicing. It is unclear how different exons are positioned precisely with U5 loop 1. Here, we investigate the role of U2 and U6 in positioning the exons with U5 loop 1. Reconstitution in vitro of spliceosomes with mutations in U2 allows U5-pre-mRNA interactions before the first step of splicing. However, insertion in U2 helix Ia disrupts U5-exon interactions with the intron lariat-3' exon splicing intermediate. Conversely, U6 helix Ia insertions prevent U5-pre-mRNA interactions before the first step of splicing. In vivo, synthetic lethal interactions have been identified between U2 insertion and U5 loop 1 insertion mutants. Additionally, analysis of U2 insertion mutants in vivo reveals that they influence the efficiency, but not the accuracy of splicing. Our data suggest that U2 aligns the exons with U5 loop 1 for ligation during the second step of pre-mRNA splicing.     

Proximity of conserved U6 and U2 snRNA elements to the 5' splice site region in activated spliceosomes

Major structural changes occur in the spliceosome during its catalytic activation, which immediately precedes the splicing of pre-mRNA. Whereas changes in snRNA conformation are well documented at the level of secondary RNA-RNA interactions, little is known about the tertiary structure of this RNA-RNA network, which comprises the spliceosome's catalytic core. Here, we have used the hydroxyl-radical probe Fe-BABE, tethered to the tenth nucleotide (U(+10)) of the 5' end of a pre-mRNA intron, to map RNA-RNA proximities in spliceosomes. These studies revealed that several conserved snRNA regions are close to U(+10) in activated spliceosomes, namely (i) the U6 snRNA ACAGAG-box region, (ii) portions of the U6 intramolecular stem-loop (U6-ISL) including a nucleotide implicated in the first catalytic step (U74), and (iii) the region of U2 that interacts with the branch point. These data constrain the relative orientation of these structural elements with respect to U(+10) in the activated spliceosome. Upon conversion of the activated spliceosome to complex C, the accessibility of U6-ISL to hydroxyl-radical cleavage is altered, suggesting rearrangements after the first catalytic step.     

U7 snRNAs: a computational survey

U7 small nuclear RNA (snRNA) sequences have been described only for a handful of animal species in the past. Here we describe a computational search for functional U7 snRNA genes throughout vertebrates including the upstream sequence elements characteristic for snRNAs transcribed by polymerase II. Based on the results of this search, we discuss the high variability of U7 snRNAs in both sequence and structure, and report on an attempt to find U7 snRNA sequences in basal deuterostomes and non-drosophilids insect genomes based on a combination of sequence, structure, and promoter features. Due to the extremely short sequence and the high variability in both sequence and structure, no unambiguous candidates were found. These results cast doubt on putative U7 homologs in even more distant organisms that are reported in the most recent release of the Rfam database.     

Molecular analysis of dicot and monocot small nuclear RNA populations.

Oligonucleotides directed against conserved small nuclear RNA (snRNA) sequences have been used to identify the individual U1, U2, U4, U5, and U6 snRNAs in dicot and monocot nuclei. The plant snRNA populations are significantly more heterogeneous than the mammalian or Saccharomyces cerevisiae snRNA populations. U6 snRNA exists as a single species of similar size in monocot and dicot nuclei. The abundance and molecular weights of the U1, U2, U4, and U5 snRNAs expressed in monocot and dicot nuclei are significantly different. Whereas most dicot nuclei contain one or two predominant forms of U2 snRNA and a small number of U4 snRNAs, monocot nuclei contain multiple forms of U2 snRNA ranging from 208 to 260 nucleotides and multiple forms of U4 snRNA from 159 to 176 nucleotides. Multiple forms of U1 and U5 snRNA exist in both plant groups. All prominent size variants of U1, U2, U4, and U5 snRNA identified in monocot nuclei can be immunoprecipitated with anti-trimethylguanosine antibody. We conclude that the sizes and number of snRNA molecules involved in intron excision differ considerably in dicot and monocot nuclei. In wheat nuclei, we have identified an additional U1-like RNA that is differentially expressed during development.     

RNA structure and RNA-protein interactions in purified yeast U6 snRNPs

The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2p-8p proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal function. However, the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. Here, we have investigated the secondary structure of the U6 snRNA in purified U6 snRNPs and compared it with its naked form. Using RNA structure-probing techniques, we demonstrate that within the U6 snRNP a large internal region of the U6 snRNA is unpaired and protected from chemical modification by bound Prp24p. Several of these U6 nucleotides are available for base-pairing interaction, as only their sugar backbone is contacted by Prp24p. Thus, Prp24p can present them to the U4 snRNA and facilitate formation of U4/U6 stem I. We show that the 3' stem-loop is not bound strongly by U6 proteins in native particles. However, when compared to the 3' stem-loop in the naked U6 snRNA, it has a more open conformation, which would facilitate formation of stem II with the U4 snRNA. Our data suggest that the combined association of Prp24p and the LSm complex confers upon U6 nucleotides a conformation favourable for U4/U6 base-pairing. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs.     

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5′ splice sites at exon/intron boundaries. U1 snRNAs associate with 5′ splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the ‘Smith antigen’, or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs.