U1A Regulates 3′ Processing of the Survival Motor Neuron mRNA

Insufficient expression of the survival motor neuron (SMN) protein causes spinal muscular atrophy, a neurodegenerative disease characterized by loss of motor neurons. Despite the importance of maintaining adequate SMN levels, little is known about factors that control SMN expression, particularly 3′ end processing of the SMN pre-mRNA. In this study, we identify the U1A protein as a key regulator of SMN expression. U1A, a component of the U1 snRNP, is known to inhibit polyadenylation upon direct binding to mRNA. We show that U1A binds directly and with high affinity and specificity to the SMN 3′-UTR adjacent to the polyadenylation site, independent of the U1 snRNP (U1 small nuclear ribonucleoprotein). Binding of U1A inhibits polyadenylation of the SMN pre-mRNA by specifically inhibiting 3′ cleavage by the cleavage and polyadenylation specificity factor. Expression of U1A in excess of U1 snRNA causes inhibition of SMN polyadenylation and decreases SMN protein levels. This work reveals a new mechanism for regulating SMN levels and provides new insight into the roles of U1A in 3′ processing of mRNAs.     

Sequence-specific interaction of U1 snRNA with the SMN complex

The survival of motor neurons (SMN) protein complex functions in the biogenesis of spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and prob ably other RNPs. All spliceosomal snRNPs have a common core of seven Sm proteins. To mediate the assembly of snRNPs, the SMN complex must be able to bring together Sm proteins with U snRNAs. We showed previously that SMN and other components of the SMN complex interact directly with several Sm proteins. Here, we show that the SMN complex also interacts specifically with U1 snRNA. The stem--loop 1 domain of U1 (SL1) is necessary and sufficient for SMN complex binding in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. These findings indicate that SMN complex interaction with SL1 is sequence-specific and critical for U1 snRNP biogenesis, further supporting the direct role of the SMN complex in RNP biogenesis.     

The Pre-mRNA 5′ Cap Determines Whether U6 Small Nuclear RNA Succeeds U1 Small Nuclear Ribonucleoprotein Particle at 5′ Splice Sites

Efficient splicing of the 5′-most intron of pre-mRNA requires a 5′ m⁷G(5′)ppp(5′)N cap, which has been implicated in U1 snRNP binding to 5′ splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5′ cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5′ splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5′ splice site and not with any loss of U1 snRNP binding.     

Ongoing U snRNP biogenesis is required for the integrity of Cajal bodies

Cajal bodies (CBs) have been implicated in the nuclear phase of the biogenesis of spliceosomal U small nuclear ribonucleoproteins (U snRNPs). Here, we have investigated the distribution of the CB marker protein coilin, U snRNPs, and proteins present in C/D box small nucleolar (sno)RNPs in cells depleted of hTGS1, SMN, or PHAX. Knockdown of any of these three proteins by RNAi interferes with U snRNP maturation before the reentry of U snRNA Sm cores into the nucleus. Strikingly, CBs are lost in the absence of hTGS1, SMN, or PHAX and coilin is dispersed in the nucleoplasm into numerous small foci. This indicates that the integrity of canonical CBs is dependent on ongoing U snRNP biogenesis. Spliceosomal U snRNPs show no detectable concentration in nuclear foci and do not colocalize with coilin in cells lacking hTGS1, SMN, or PHAX. In contrast, C/D box snoRNP components concentrate into nuclear foci that partially colocalize with coilin after inhibition of U snRNP maturation. We demonstrate by siRNA-mediated depletion that coilin is required for the condensation of U snRNPs, but not C/D box snoRNP components, into nucleoplasmic foci, and also for merging these factors into canonical CBs. Altogether, our data suggest that CBs have a modular structure with distinct domains for spliceosomal U snRNPs and snoRNPs.     

pICln Inhibits snRNP Biogenesis by Binding Core Spliceosomal Proteins

The U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) form essential components of spliceosomes, the machinery that removes introns from pre-mRNAs in eukaryotic cells. A critical initial step in the complex process of snRNP biogenesis is the assembly of a group of common core proteins (Sm proteins) on spliceosomal snRNA. In this study we show by multiple independent methods that the protein pICln associates with Sm proteins in vivo and in vitro. The binding of pICln to Sm proteins interferes with Sm protein assembly on spliceosomal snRNAs and inhibits import of snRNAs into the nucleus. Furthermore, pICln prevents the interaction of Sm proteins with the survival of motor neurons (SMN) protein, an interaction that has been shown to be critical for snRNP biogenesis. These findings lead us to propose a model in which pICln participates in the regulation of snRNP biogenesis, at least in part by interfering with Sm protein interaction with SMN protein.     

Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation

Neuronal degeneration in spinal muscular atrophy (SMA) is caused by reduced expression of the survival of motor neuron (SMN) protein. The SMN protein is ubiquitously expressed and is present both in the cytoplasm and in the nucleus where it localizes in Cajal bodies. The SMN complex plays an essential role for the biogenesis of spliceosomal U-snRNPs. In this article, we have used an RNA interference approach in order to analyse the effects of SMN depletion on snRNP assembly in HeLa cells. Although snRNP profiles are not perturbed in SMN-depleted cells, we found that SMN depletion gives rise to cytoplasmic accumulation of a GFP-SmB reporter protein. We also demonstrate that the SMN protein depletion induces defects in Cajal body formation with coilin being localized in multiple nuclear foci and in nucleolus instead of canonical Cajal bodies. Interestingly, the coilin containing foci do not contain snRNPs but appear to co-localize with U85 scaRNA. Because Cajal bodies represent the location in which snRNPs undergo 2′-O-methylation and pseudouridylation, our results raise the possibility that SMN depletion might give rise to a defect in the snRNA modification process.     

Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein,survival of motor neuron

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm-core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5′ cap. Both the Sm-core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs. Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein. Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies. The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy. These data indicate that, in addition to its function in cytoplasmic Sm-core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.     

snRNAs contain specific SMN-binding domains that are essential for snRNP assembly

To serve in its function as an assembly machine for spliceosomal small nuclear ribonucleoprotein particles (snRNPs), the survival of motor neurons (SMN) protein complex binds directly to the Sm proteins and the U snRNAs. A specific domain unique to U1 snRNA, stem-loop 1 (SL1), is required for SMN complex binding and U1 snRNP Sm core assembly. Here, we show that each of the major spliceosomal U snRNAs (U2, U4, and U5), as well as the minor splicing pathway U11 snRNA, contains a domain to which the SMN complex binds directly and with remarkable affinity (low nanomolar concentration). The SMN-binding domains of the U snRNAs do not have any significant nucleotide sequence similarity yet they compete for binding to the SMN complex in a manner that suggests the presence of at least two binding sites. Furthermore, the SMN complex-binding domain and the Sm site are both necessary and sufficient for Sm core assembly and their relative positions are critical for snRNP assembly. These findings indicate that the SMN complex stringently scrutinizes RNAs for specific structural features that are not obvious from the sequence of the RNAs but are required for their identification as bona fide snRNAs. It is likely that this surveillance capacity of the SMN complex ensures assembly of Sm cores on the correct RNAs only and prevents illicit, potentially deleterious, assembly of Sm cores on random RNAs.     

Why do cells need an assembly machine for RNA-protein complexes?

Small nuclear ribonucleoproteins (snRNPs) are crucial for pre-mRNA processing to mRNAs. Each snRNP contains a small nuclear RNA (snRNA) and an extremely stable core of seven Sm proteins. The snRNP biogenesis pathway is complex, involving nuclear export of snRNA, Sm-core assembly in the cytoplasm and re-import of the mature snRNP. Although in vitro Sm cores assemble readily on uridine-rich RNAs, the assembly in cells is carried out by the survival of motor neurons (SMN) complex. The SMN complex stringently scrutinizes RNAs for specific features that define them as snRNAs and identifies the RNA-binding Sm proteins. We discuss how this surveillance capacity of the SMN complex might ensure assembly of Sm cores only on the correct RNAs and prevent illicit, potentially deleterious assembly of Sm cores on random RNAs.     

A novel EB-1/AIDA-1 isoform,AIDA-1c,interacts with the Cajal body protein coilin

Background  

Cajal bodies (CBs) are nuclear suborganelles that play a role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are crucial for pre-mRNA splicing. Upon nuclear reentry, Sm-class snRNPs localize first to the CB, where the snRNA moiety of the snRNP is modified. It is not clear how snRNPs target to the CB and are released from this structure after their modification. Coilin, the CB marker protein, may participate in snRNP biogenesis given that it can interact with snRNPs and SMN. SMN is crucial for snRNP assembly and is the protein mutated in the neurodegenerative disease Spinal Muscular Atrophy. Coilin knockout mice display significant viability problems and altered CB formation. Thus characterization of the CB and its associated proteins will give insight into snRNP biogenesis and clarify the dynamic organization of the nucleus.     

The SMN complex is associated with snRNPs throughout their cytoplasmic assembly pathway

The common neurodegenerative disease spinal muscular atrophy is caused by reduced levels of the survival of motor neurons (SMN) protein. SMN associates with several proteins (Gemin2 to Gemin6) to form a large complex which is found both in the cytoplasm and in the nucleus. The SMN complex functions in the assembly and metabolism of several RNPs, including spliceosomal snRNPs. The snRNP core assembly takes place in the cytoplasm from Sm proteins and newly exported snRNAs. Here, we identify three distinct cytoplasmic SMN complexes, each representing a defined intermediate in the snRNP biogenesis pathway. We show that the SMN complex associates with newly exported snRNAs containing the nonphosphorylated form of the snRNA export factor PHAX. The second SMN complex identified contains assembled Sm cores and m(3)G-capped snRNAs. Finally, the SMN complex is associated with a preimport complex containing m(3)G-capped snRNP cores bound to the snRNP nuclear import mediator snurportin1. Thus, the SMN complex is associated with snRNPs during the entire process of their biogenesis in the cytoplasm and may have multiple functions throughout this process.     

Exon‐independent recruitment of SRSF1 is mediated by U1 snRNP stem‐loop 3

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5′ splice site (SS), and both factors affect 5′ SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single‐molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1‐independent process of binding to an ESE. Structural analysis and cross‐linking data show that SRSF1 contacts U1 snRNA stem‐loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5′SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.     

Binding of human SLBP on the 3'-UTR of histone precursor H4-12 mRNA induces structural rearrangements that enable U7 snRNA anchoring

In metazoans, cell-cycle-dependent histones are produced from poly(A)-lacking mRNAs. The 3′ end of histone mRNAs is formed by an endonucleolytic cleavage of longer precursors between a conserved stem–loop structure and a purine-rich histone downstream element (HDE). The cleavage requires at least two trans-acting factors: the stem–loop binding protein (SLBP), which binds to the stem–loop and the U7 snRNP, which anchors to histone pre-mRNAs by annealing to the HDE. Using RNA structure-probing techniques, we determined the secondary structure of the 3′-untranslated region (3′-UTR) of mouse histone pre-mRNAs H4–12, H1t and H2a–614. Surprisingly, the HDE is embedded in hairpin structures and is therefore not easily accessible for U7 snRNP anchoring. Probing of the 3′-UTR in complex with SLBP revealed structural rearrangements leading to an overall opening of the structure especially at the level of the HDE. Electrophoretic mobility shift assays demonstrated that the SLBP-induced opening of HDE actually facilitates U7 snRNA anchoring on the histone H4–12 pre-mRNAs 3′ end. These results suggest that initial binding of the SLBP functions in making the HDE more accessible for U7 snRNA anchoring.     

The U1, U2 and U5 snRNAs crosslink to the 5' exon during yeast pre-mRNA splicing

Activation of pre-messenger RNA (pre-mRNA) splicing requires 5′ splice site recognition by U1 small nuclear RNA (snRNA), which is replaced by U5 and U6 snRNA. Here we use crosslinking to investigate snRNA interactions with the 5′ exon adjacent to the 5′ splice site, prior to the first step of splicing. U1 snRNA was found to interact with four different 5′ exon positions using one specific sequence adjacent to U1 snRNA helix 1. This novel interaction of U1 we propose occurs before U1-5′ splice site base pairing. In contrast, U5 snRNA interactions with the 5′ exon of the pre-mRNA progressively shift towards the 5′ end of U5 loop 1 as the crosslinking group is placed further from the 5′ splice site, with only interactions closest to the 5′ splice site persisting to the 5′ exon intermediate and the second step of splicing. A novel yeast U2 snRNA interaction with the 5′ exon was also identified, which is ATP dependent and requires U2-branchpoint interaction. This study provides insight into the nature and timing of snRNA interactions required for 5′ splice site recognition prior to the first step of pre-mRNA splicing.     

Functional studies on the ATM intronic splicing processing element

In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5′ and 3′ splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a ~40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5′–3′ order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.     

A Drosophila model of spinal muscular atrophy uncouples snRNP biogenesis functions of survival motor neuron from locomotion and viability defects

The spinal muscular atrophy (SMA) protein, survival motor neuron (SMN), functions in the biogenesis of small nuclear ribonucleoproteins (snRNPs). SMN has also been implicated in tissue-specific functions; however, it remains unclear which of these is important for the etiology of SMA. Smn null mutants display larval lethality and show significant locomotion defects as well as reductions in minor-class spliceosomal snRNAs. Despite these reductions, we found no appreciable defects in the splicing of mRNAs containing minor-class introns. Transgenic expression of low levels of either wild-type or an SMA patient-derived form of SMN rescued the larval lethality and locomotor defects; however, snRNA levels were not restored. Thus, the snRNP biogenesis function of SMN is not a major contributor to the phenotype of Smn null mutants. These findings have major implications for SMA etiology because they show that SMN's role in snRNP biogenesis can be uncoupled from the organismal viability and locomotor defects.