Splicing-Correcting Therapy for SMA.

Spinal muscular atrophy (SMA) is caused by deficiency of SMN protein, which is crucial for spliceosome subunits biogenesis. Most SMA patients have SMN1 deletions, leaving SMN2 as sole SMN source; however, a C→T substitution converts an exonic-splicing enhancer (ESE) to a silencer (ESS), causing frequent exon7 skipping in SMN2 pre-mRNA and yielding a truncated protein. Antisense treatment to SMN2 intron7-splicing silencer (ISS) improves SMN expression and motor function. To view this Bench to Bedside, open or download the PDF.

U1 snRNP determines mRNA length and regulates isoform expression.

U1 snRNP (U1), in addition to its splicing role, protects pre-mRNAs from drastic premature termination by cleavage and polyadenylation (PCPA) at cryptic polyadenylation signals (PASs) in introns. Here, a high-throughput sequencing strategy of differentially expressed transcripts (HIDE-seq) mapped PCPA sites genome wide in divergent organisms. Surprisingly, whereas U1 depletion terminated most nascent gene transcripts within ~1 kb, moderate functional U1 level decreases, insufficient to inhibit splicing, dose-dependently shifted PCPA downstream and elicited mRNA 3' UTR shortening and proximal 3' exon switching characteristic of activated immune and neuronal cells, stem cells, and cancer. Activated neurons' signature mRNA shortening could be recapitulated by U1 decrease and antagonized by U1 overexpression. Importantly, we show that rapid and transient transcriptional upregulation inherent to neuronal activation physiology creates U1 shortage relative to pre-mRNAs. Additional experiments suggest cotranscriptional PCPA counteracted by U1 association with nascent transcripts, a process we term telescripting, ensuring transcriptome integrity and regulating mRNA length.

A Complex of U1 snRNP with Cleavage and Polyadenylation Factors Controls Telescripting, Regulating mRNA Transcription in Human Cells.

Full-length transcription in the majority of human genes depends on U1 snRNP (U1) to co-transcriptionally suppress transcription-terminating premature 3' end cleavage and polyadenylation (PCPA) from cryptic polyadenylation signals (PASs) in introns. However, the mechanism of this U1 activity, termed telescripting, is unknown. Here, we captured a complex, comprising U1 and CPA factors (U1-CPAFs), that binds intronic PASs and suppresses PCPA. U1-CPAFs are distinct from U1-spliceosomal complexes; they include CPA's three main subunits, CFIm, CPSF, and CstF; lack essential splicing factors; and associate with transcription elongation and mRNA export complexes. Telescripting requires U1:pre-mRNA base-pairing, which can be disrupted by U1 antisense oligonucleotide (U1 AMO), triggering PCPA. U1 AMO remodels U1-CPAFs, revealing changes, including recruitment of CPA-stimulating factors, that explain U1-CPAFs' switch from repressive to activated states. Our findings outline this U1 telescripting mechanism and demonstrate U1's unique role as central regulator of pre-mRNA processing and transcription.

Structure of a key intermediate of the SMN complex reveals Gemin2's crucial function in snRNP assembly.

The SMN complex mediates the assembly of heptameric Sm protein rings on small nuclear RNAs (snRNAs), which are essential for snRNP function. Specific Sm core assembly depends on Sm proteins and snRNA recognition by SMN/Gemin2- and Gemin5-containing subunits, respectively. The mechanism by which the Sm proteins are gathered while preventing illicit Sm assembly on non-snRNAs is unknown. Here, we describe the 2.5 Å crystal structure of Gemin2 bound to SmD1/D2/F/E/G pentamer and SMN's Gemin2-binding domain, a key assembly intermediate. Remarkably, through its extended conformation, Gemin2 wraps around the crescent-shaped pentamer, interacting with all five Sm proteins, and gripping its bottom and top sides and outer perimeter. Gemin2 reaches into the RNA-binding pocket, preventing RNA binding. Interestingly, SMN-Gemin2 interaction is abrogated by a spinal muscular atrophy (SMA)-causing mutation in an SMN helix that mediates Gemin2 binding. These findings provide insight into SMN complex assembly and specificity, linking snRNP biogenesis and SMA pathogenesis.

RNA and disease.

Cellular functions depend on numerous protein-coding and noncoding RNAs and the RNA-binding proteins associated with them, which form ribonucleoprotein complexes (RNPs). Mutations that disrupt either the RNA or protein components of RNPs or the factors required for their assembly can be deleterious. Alternative splicing provides cells with an exquisite capacity to fine-tune their transcriptome and proteome in response to cues. Splicing depends on a complex code, numerous RNA-binding proteins, and an enormously intricate network of interactions among them, increasing the opportunity for exposure to mutations and misregulation that cause disease. The discovery of disease-causing mutations in RNAs is yielding a wealth of new therapeutic targets, and the growing understanding of RNA biology and chemistry is providing new RNA-based tools for developing therapeutics.

Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.

Here we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation.

SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing.

The survival of motor neurons (SMN) protein is essential for the biogenesis of small nuclear RNA (snRNA)-ribonucleoproteins (snRNPs), the major components of the pre-mRNA splicing machinery. Though it is ubiquitously expressed, SMN deficiency causes the motor neuron degenerative disease spinal muscular atrophy (SMA). We show here that SMN deficiency, similar to that which occurs in severe SMA, has unexpected cell type-specific effects on the repertoire of snRNAs and mRNAs. It alters the stoichiometry of snRNAs and causes widespread pre-mRNA splicing defects in numerous transcripts of diverse genes, preferentially those containing a large number of introns, in SMN-deficient mouse tissues. These findings reveal a key role for the SMN complex in RNA metabolism and in splicing regulation and indicate that SMA is a general splicing disease that is not restricted to motor neurons.

Ars2 promotes proper replication-dependent histone mRNA 3' end formation.

Ars2 is a component of the nuclear cap-binding complex that contributes to microRNA biogenesis and is required for cellular proliferation. Here, we expand on the repertoire of Ars2-dependent microRNAs and determine that Ars2 regulates a number of mRNAs, the largest defined subset of which code for histones. Histone mRNAs are unique among mammalian mRNAs because they are not normally polyadenylated but, rather, are cleaved following a 3' stem loop. A significant reduction in correctly processed histone mRNAs was observed following Ars2 depletion, concurrent with an increase in polyadenylated histone transcripts. Furthermore, Ars2 physically associated with histone mRNAs and the noncoding RNA 7SK. Knockdown of 7SK led to an enhanced ratio of cleaved to polyadenylated histone transcripts, an effect dependent on Ars2. Together, the data demonstrate that Ars2 contributes to histone mRNA 3' end formation and expression and these functional properties of Ars2 are negatively regulated by interaction with 7SK RNA.

Gemin5 delivers snRNA precursors to the SMN complex for snRNP biogenesis.

The SMN complex assembles Sm cores on snRNAs, a key step in the biogenesis of snRNPs, the spliceosome's major components. Here, using SMN complex inhibitors identified by high-throughput screening and a ribo-proteomic strategy on formaldehyde crosslinked RNPs, we dissected this pathway in cells. We show that protein synthesis inhibition impairs the SMN complex, revealing discrete SMN and Gemin subunits and accumulating an snRNA precursor (pre-snRNA)-Gemin5 intermediate. By high-throughput sequencing of this transient intermediate's RNAs, we discovered the previously undetectable precursors of all the snRNAs and identified their Gemin5-binding sites. We demonstrate that pre-snRNA 3' sequences function to enhance snRNP biogenesis. The SMN complex is also inhibited by oxidation, and we show that it stalls an inventory-complete SMN complex containing pre-snRNAs. We propose a stepwise pathway of SMN complex formation and snRNP biogenesis, highlighting Gemin5's function in delivering pre-snRNAs as substrates for Sm core assembly and processing.

tRNA binds to cytochrome c and inhibits caspase activation.

The specific molecular events that characterize the intrinsic apoptosis pathway have been the subject of intense research due to the pathway's fundamental role in development, homeostasis, and cancer. This pathway is defined by the release of cytochrome c from mitochondria into the cytosol and subsequent binding of cytochrome c to the caspase activator Apaf-1. Here, we report that both mitochondrial and cytosolic transfer RNA (tRNA) bind to cytochrome c. This binding prevents cytochrome c interaction with Apaf-1, blocking Apaf-1 oligomerization and caspase activation. tRNA hydrolysis in living cells and cell lysates enhances apoptosis and caspase activation, whereas microinjection of tRNA into living cells blocks apoptosis. These findings suggest that tRNA, in addition to its well-established role in gene expression, may determine cellular responsiveness to apoptotic stimuli.

A degron created by SMN2 exon 7 skipping is a principal contributor to spinal muscular atrophy severity.

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletions, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, most of the mRNA produced from SMN2 pre-mRNA is exon 7-skipped ( approximately 80%), resulting in a highly unstable and almost undetectable protein (SMNDelta7). We show that this splicing defect creates a potent degradation signal (degron; SMNDelta7-DEG) at SMNDelta7's C-terminal 15 amino acids. The S270A mutation inactivates SMNDelta7-DEG, generating a stable SMNDelta7 that rescues viability of SMN-deleted cells. These findings explain a key aspect of the SMA disease mechanism, and suggest new treatment approaches based on interference with SMNDelta7-DEG activity.

U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation.

In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA-pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

Inactivation of the SMN complex by oxidative stress.

The SMN complex is essential for the biogenesis of small nuclear ribonucleoproteins (snRNPs), the major constituents of the spliceosome. Deficiency in functional SMN protein causes spinal muscular atrophy, a common motor neuron degenerative disease of severity commensurate with SMN levels and, correspondingly, snRNP assembly decreases. We developed a high-throughput screen for snRNP assembly modifiers and discovered that reactive oxygen species (ROS) inhibit SMN-complex activity in a dose-dependent manner. ROS-generating compounds, e.g., the environmental toxins menadione and beta-lapachone (in vivo IC(50) = 0.45 muM) also cause intermolecular disulfide crosslinking of SMN. Both the oxidative inactivation and SMN crosslinking can be reversed by reductants. We identified two cysteines that form SMN-SMN disulfide crosslinks, defining specific contact points in oligomeric SMN. Thus, the SMN complex is a redox-sensitive assemblyosome and an ROS target, suggesting that it may play a role in oxidative stress pathophysiology, which is associated with many degenerative diseases.

Dysregulation of synaptogenesis genes antecedes motor neuron pathology in spinal muscular atrophy.

The motor neuron (MN) degenerative disease, spinal muscular atrophy (SMA) is caused by deficiency of SMN (survival motor neuron), a ubiquitous and indispensable protein essential for biogenesis of snRNPs, key components of pre-mRNA processing. However, SMA's hallmark MN pathology, including neuromuscular junction (NMJ) disruption and sensory-motor circuitry impairment, remains unexplained. Toward this end, we used deep RNA sequencing (RNA-seq) to determine if there are any transcriptome changes in MNs and surrounding spinal cord glial cells (white matter, WM) microdissected from SMN-deficient SMA mouse model at presymptomatic postnatal day 1 (P1), before detectable MN pathology (P4-P5). The RNA-seq results, previously unavailable for SMA at any stage, revealed cell-specific selective mRNA dysregulations (~300 of 11,000 expressed genes in each, MN and WM), many of which are known to impair neurons. Remarkably, these dysregulations include complete skipping of agrin's Z exons, critical for NMJ maintenance, strong up-regulation of synapse pruning-promoting complement factor C1q, and down-regulation of Etv1/ER81, a transcription factor required for establishing sensory-motor circuitry. We propose that dysregulation of such specific MN synaptogenesis genes, compounded by many additional transcriptome abnormalities in MNs and WM, link SMN deficiency to SMA's signature pathology.

Genomics-guided discovery of thailanstatins A, B, and C As pre-mRNA splicing inhibitors and antiproliferative agents from Burkholderia thailandensis MSMB43.

Mining the genome sequence of Burkholderia thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster resembling that of FR901464 (4), a prototype spliceosome inhibitor produced by Pseudomonas sp. No. 2663. Transcriptional analysis revealed a cultivation condition in which a regulatory gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, named thailanstatins A (1), B (2), and C (3), were isolated from the fermentation broth of B. thailandensis MSMB43. Thailanstatins are proposed to be biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase pathway. They differ from 4 by lacking an unstable hydroxyl group and by having an extra carboxyl moiety; those differences endow thailanstatins with a significantly greater stability than 4 as tested in phosphate buffer at pH 7.4. In vitro assays showed that thailanstatins inhibit pre-mRNA splicing as potently as 4, with half-maximal inhibitory concentrations in the single to sub-µM range. Cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range. This work provides new chemical entities for research and development and new structure-activity information for chemical optimization of related spliceosome inhibitors.

The SMN complex, an assemblyosome of ribonucleoproteins.

Spinal muscular atrophy is a common, often lethal, neurodegenerative disease that results from low levels of, or loss-of-function mutations in, the SMN (survival of motor neurons) protein. SMN oligomerizes and forms a stable complex with five additional proteins: Gemins 2-6. SMN also interacts with several additional proteins referred to as "substrates". Most of these substrates contain a domain enriched in arginine and glycine residues (the RG-rich domain), and are constituents of different ribonucleoprotein complexes. Recent studies revealed that the substrates can be modified by an arginine methyltransferase complex, the methylosome. This forms symmetrical dimethylarginines within the RG-rich domains of the substrates, thereby converting them to high-affinity binders of the SMN complex, and most likely providing regulation of the ribonucleoprotein assembly processes.

Comprehensive RNP profiling in cells identifies U1 snRNP complexes with cleavage and polyadenylation factors active in telescripting.

Full-length transcription in the majority of protein-coding and other genes transcribed by RNA polymerase II in complex eukaryotes requires U1 snRNP (U1) to co-transcriptionally suppress transcription-terminating premature 3'-end cleavage and polyadenylation (PCPA) from cryptic polyadenylation signals (PASs). This U1 activity, termed telescripting, requires U1 to base-pair with the nascent RNA and inhibit usage of a downstream PAS. Here we describe experimental methods to determine the mechanism of U1 telescripting, involving mapping of U1 and CPA factors (CPAFs) binding locations in relation to PCPA sites, and identify U1 and CPAFs interactomes. The methods which utilizes rapid reversible protein-RNA and protein-protein chemical crosslinking, immunoprecipitations (XLIPs) of components of interest, and RNA-seq and quantitative proteomic mass spectrometry, captured U1-CPAFs complexes in cells, providing important insights into telescripting mechanism. XLIP profiling can be used for comprehensive molecular definition of diverse RNPs.

U1 snRNP regulates cancer cell migration and invasion in vitro.

Stimulated cells and cancer cells have widespread shortening of mRNA 3'-untranslated regions (3'UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in introns and last exons. U1 snRNP (U1), vertebrates' most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread premature transcription termination and mRNA shortening. Here we show that low U1 AMO doses increase cancer cells' migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect. In addition to 3'UTR length, numerous transcriptome changes that could contribute to this phenotype are observed, including alternative splicing, and mRNA expression levels of proto-oncogenes and tumor suppressors. These findings reveal an unexpected role for U1 homeostasis (available U1 relative to transcription) in oncogenic and activated cell states, and suggest U1 as a potential target for their modulation.

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.