Extended base pair complementarity between U1 snRNA and the 5' splice site does not inhibit splicing in higher eukaryotes, but rather increases 5' splice site recognition

Spliceosome formation is initiated by the recognition of the 5′ splice site through formation of an RNA duplex between the 5′ splice site and U1 snRNA. We have previously shown that RNA duplex formation between U1 snRNA and the 5′ splice site can protect pre-mRNAs from degradation prior to splicing. This initial RNA duplex must be disrupted to expose the 5′ splice site sequence for base pairing with U6 snRNA and to form the active spliceosome. Here, we investigated whether hyperstabilization of the U1 snRNA/5′ splice site duplex interferes with splicing efficiency in human cell lines or nuclear extracts. Unlike observations in Saccharomyces cerevisiae, we demonstrate that an extended U1 snRNA/5′ splice site interaction does not decrease splicing efficiency, but rather increases 5′ splice site recognition and exon inclusion. However, low complementarity of the 5′ splice site to U1 snRNA significantly increases exon skipping and RNA degradation. Although the splicing mechanisms are conserved between human and S.cerevisiae, these results demonstrate that distinct differences exist in the activation of the spliceosome.     

The Exon Splicing Silencer in Human Immunodeficiency Virus Type 1 Tat Exon 3 Is Bipartite and Acts Early in Spliceosome Assembly

Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3′ splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3′ splice site together act to inhibit splicing at the 3′ splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3′ splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3′ splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.     

Structure and functional implications of a complex containing a segment of U6 RNA bound by a domain of Prp24

U6 RNA plays a critical role in pre-mRNA splicing. Assembly of U6 into the spliceosome requires a significant structural rearrangement and base-pairing with U4 RNA. In the yeast Saccharomyces cerevisiae, this process requires the essential splicing factor Prp24. We present the characterization and structure of a complex containing one of Prp24''s four RNA recognition motif (RRM) domains, RRM2, and a fragment of U6 RNA. NMR methods were used to identify the preferred U6 binding sequence of RRM2 (5′-GAGA-3′), measure the affinity of the interaction, and solve the structure of RRM2 bound to the hexaribonucleotide AGAGAU. Interdomain contacts observed between RRM2 and RRM3 in a crystal structure of the free protein are not detectable in solution. A structural model of RRM1 and RRM2 bound to a longer segment of U6 RNA is presented, and a partial mechanism for Prp24''s annealing activity is proposed.     

Alternative splicing and bioinformatic analysis of human U12-type introns

U12-type introns exist, albeit rarely, in a variety of multicellular organisms. Splicing of U12 intron-containing precursor mRNAs takes place in the U12-type spliceosome that is distinct from the major U2-type spliceosome. Due to incompatibility of these two spliceosomes, alternative splicing involving a U12-type intron may give rise to a relatively complicated impact on gene expression. We studied alternative U12-type intron splicing in an attempt to gain more mechanistic insights. First, we characterized mutually exclusive exon selection of the human JNK2 gene, which involves an unusual intron possessing the U12-type 5′ splice site and the U2-type 3′ splice site. We demonstrated that the long and evolutionary conserved polypyrimidine tract of this hybrid intron provides important signals for inclusion of its downstream alternative exon. In addition, we examined the effects of single nucleotide polymorphisms in the human WDFY1 U12-type intron on pre-mRNA splicing. These results provide mechanistic implications on splice-site selection of U12-type intron splicing. We finally discuss the potential effects of splicing of a U12-type intron with genetic defects or within a set of genes encoding RNA processing factors on global gene expression.     

Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs

Saccharomyces cerevisiae Msl5 orchestrates spliceosome assembly by binding the intron branchpoint sequence 5′-UACUAAC and, with its heterodimer partner protein Mud2, establishing cross intron-bridging interactions with the U1 snRNP at the 5′ splice site. Here we define the central Msl5 KH-QUA2 domain as sufficient for branchpoint RNA recognition. The 1.8 Å crystal structure of Msl5-(KH-QUA2) bound to the branchpoint highlights an extensive network of direct and water-mediated protein–RNA and intra-RNA atomic contacts at the interface that illuminate how Msl5 recognizes each nucleobase of the UACUAAC element. The Msl5 structure rationalizes a large body of mutational data and inspires new functional studies herein, which reveal how perturbations of the Msl5·RNA interface impede the splicing of specific yeast pre-mRNAs. We also identify interfacial mutations in Msl5 that bypass the essentiality of Sub2, a DExD-box ATPase implicated in displacing Msl5 from the branchpoint in exchange for the U2 snRNP. These studies establish an atomic resolution framework for understanding splice site selection and early spliceosome dynamics.     

Insights into the activation of the helicase Prp43 by biochemical studies and structural mass spectrometry

Splicing of precursor messenger RNA is a hallmark of eukaryotic cells, which is carried out by the spliceosome, a multi-megadalton ribonucleoprotein machinery. The splicing reaction removes non-coding regions (introns) and ligates coding regions (exons). The spliceosome is a highly dynamic ribonucleoprotein complex that undergoes dramatic structural changes during its assembly, the catalysis and its disassembly. The transitions between the different steps during the splicing cycle are promoted by eight conserved DExD/H box ATPases. The DEAH-box protein Prp43 is responsible for the disassembly of the intron-lariat spliceosome and its helicase activity is activated by the G-patch protein Ntr1. Here, we investigate the activation of Prp43 by Ntr1 in the presence and absence of RNA substrate by functional assays and structural proteomics. Residues 51–110 of Ntr1 were identified to be the minimal fragment that induces full activation. We found protein–protein cross-links that indicate that Prp43 interacts with the G-patch motif of Ntr1 through its C-terminal domains. Additionally, we report on functionally important RNA binding residues in both proteins and propose a model for the activation of the helicase.     

The Interaction of Prp2 with a Defined Region of the Intron Is Required for the First Splicing Reaction

In Saccharomyces cerevisiae, the 3′ splice site is not required for the first catalytic reaction of splicing. We have previously reported that at least 24 nucleotides downstream of the branch point is required for the first reaction to take place, but the precatalytic spliceosome forms efficiently on the truncated pre-mRNA with only 5 nucleotides retained downstream of the branch point. The factors that mediate this length-dependent control of the first catalytic step are not known. We show here that Prp2 can be recruited to the spliceosome without interacting with pre-mRNA when the 3′ tail is short. Prp2 interacts with the intron when the 3′ tail is extended, which results in destabilization of Prp2 and, consequently, progression of the first reaction. An RNA segment at 23 to 33 nucleotides downstream of the branch point is necessary and sufficient for the ATP-dependent action of Prp2. We also show that Prp2 directly interacts with the carboxyl-terminal fragment of Brr2 by pulldown assays. We propose that Prp2 is recruited to the spliceosome via interaction with Brr2 and is spatially positioned to interact with this specific region of the pre-mRNA, which stimulates the ATPase activity of Prp2 to promote the progression of the first catalytic step.     

The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing

Sad1 is an essential splicing factor initially identified in a genetic screen in Saccharomyces cerevisiae for snRNP assembly defects. Based on sequence homology, Sad1, or USP39 in humans, is predicted to comprise two domains: a zinc finger ubiquitin binding domain (ZnF-UBP) and an inactive ubiquitin-specific protease (iUSP) domain, both of which are well conserved. The role of these domains in splicing and their interaction with ubiquitin are unknown. We first used splicing microarrays to analyze Sad1 function in vivo and found that Sad1 is critical for the splicing of nearly all yeast intron-containing genes. By using in vitro assays, we then showed that it is required for the assembly of the active spliceosome. To gain structural insights into Sad1 function, we determined the crystal structure of the full-length protein at 1.8 Å resolution. In the structure, the iUSP domain forms the characteristic ubiquitin binding pocket, though with an amino acid substitution in the active site that results in complete inactivation of the enzymatic activity of the domain. The ZnF-UBP domain of Sad1 shares high structural similarly to other ZnF-UBPs; however, Sad1''s ZnF-UBP does not possess the canonical ubiquitin binding motif. Given the precedents for ZnF-UBP domains to function as activators for their neighboring USP domains, we propose that Sad1''s ZnF-UBP acts in a ubiquitin-independent capacity to recruit and/or activate Sad1''s iUSP domain to interact with the spliceosome.     

Cwc21p promotes the second step conformation of the spliceosome and modulates 3′ splice site selection

Pre-mRNA splicing involves two transesterification steps catalyzed by the spliceosome. How RNA substrates are positioned in each step and the molecular rearrangements involved, remain obscure. Here, we show that mutations in PRP16, PRP8, SNU114 and the U5 snRNA that affect this process interact genetically with CWC21, that encodes the yeast orthologue of the human SR protein, SRm300/SRRM2. Our microarray analysis shows changes in 3′ splice site selection at elevated temperature in a subset of introns in cwc21Δ cells. Considering all the available data, we propose a role for Cwc21p positioning the 3′ splice site at the transition to the second step conformation of the spliceosome, mediated through its interactions with the U5 snRNP. This suggests a mechanism whereby SRm300/SRRM2, might influence splice site selection in human cells.     

Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system

Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5′ and 3′ exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3′ splice site (3′SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3′SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.     

Detection of a Novel ATP-Dependent Cross-Linked Protein at the 5′ Splice Site-U1 Small Nuclear RNA Duplex by Methylene Blue-Mediated Photo-Cross-Linking

Assembly of spliceosomes involves a number of sequential steps in which small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP proteins recognize the splice site sequences and undergo various conformational rearrangements. A number of important intermolecular RNA-RNA duplexes are formed transiently during the process of splice site recognition. Various steps in the assembly pathway are dependent upon ATP hydrolysis, either for protein phosphorylation or for the activity of helicases, which may modulate the RNA structures. Major efforts have been made to identify proteins that interact with specific regions of the pre-mRNA during the stages of spliceosome assembly and catalysis by site-specific UV cross-linking. However, UV cross-linking is often inefficient for the detection of proteins that interact with base-paired RNA. Here we have used the complementary approach of methylene blue-mediated photo-cross-linking to detect specifically proteins that interact with the duplexes formed between pre-mRNA and small nuclear RNA (snRNA). We have detected a novel cross-link between a 65-kDa protein (p65) and the 5′ splice site. A range of data suggest that p65 cross-links to the transient duplex formed by U1 snRNA and the 5′ splice site. Moreover, although p65 cross-linking requires only a 5′ splice site within the pre-mRNA, it also requires ATP hydrolysis, suggesting that its detection reflects a very early ATP-dependent event during splicing.     

Lentivirus mediated silencing of Ubiquitin Specific Peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro

Background

Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells.

Results

In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G₂/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells.

Conclusions

All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0006-y) contains supplementary material, which is available to authorized users.     

Limited complementarity between U1 snRNA and a retroviral 5′ splice site permits its attenuation via RNA secondary structure

Multiple types of regulation are used by cells and viruses to control alternative splicing. In murine leukemia virus, accessibility of the 5′ splice site (ss) is regulated by an upstream region, which can fold into a complex RNA stem–loop structure. The underlying sequence of the structure itself is negligible, since most of it could be functionally replaced by a simple heterologous RNA stem–loop preserving the wild-type splicing pattern. Increasing the RNA duplex formation between U1 snRNA and the 5′ss by a compensatory mutation in position +6 led to enhanced splicing. Interestingly, this mutation affects splicing only in the context of the secondary structure, arguing for a dynamic interplay between structure and primary 5′ss sequence. The reduced 5′ss accessibility could also be counteracted by recruiting a splicing enhancer domain via a modified MS2 phage coat protein to a single binding site at the tip of the simple RNA stem–loop. The mechanism of 5′ss attenuation was revealed using hyperstable U1 snRNA mutants, showing that restricted U1 snRNP access is the cause of retroviral alternative splicing.     

Rearrangements within human spliceosomes captured after exon ligation

In spliceosomes, dynamic RNA/RNA and RNA/protein interactions position the pre-mRNA substrate for the two chemical steps of splicing. Not all of these interactions have been characterized, in part because it has not been possible to arrest the complex at clearly defined states relative to chemistry. Previously, it was shown in yeast that the DEAD/H-box protein Prp22 requires an extended 3′ exon to promote mRNA release from the spliceosome following second-step chemistry. In line with that observation, we find that shortening the 3′ exon blocks cleaved lariat intron and mRNA release in human splicing extracts, which allowed us to stall human spliceosomes in a new post-catalytic complex (P complex). In comparison to C complex, which is blocked at a point following first-step chemistry, we detect specific differences in RNA substrate interactions near the splice sites. These differences include extended protection across the exon junction and changes in protein crosslinks to specific sites in the 5′ and 3′ exons. Using selective reaction monitoring (SRM) mass spectrometry, we quantitatively compared P and C complex proteins and observed enrichment of SF3b components and loss of the putative RNA-dependent ATPase DHX35. Electron microscopy revealed similar structural features for both complexes. Notably, additional density is present when complexes are chemically fixed, which reconciles our results with previously reported C complex structures. Our ability to compare human spliceosomes before and after second-step chemistry has opened a new window to rearrangements near the active site of spliceosomes, which may play roles in exon ligation and mRNA release.