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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.

This summary of the 84th Cold Spring Harbor Laboratory (CSHL) Symposium on Quantitative Biology: RNA Control and Regulation, held in May 2019, highlights key emerging themes in this field, which now impacts nearly every aspect of biology and medicine. Recent discoveries accelerated by technological developments reveal enormous diversity of RNAs and RNA-binding proteins (RBPs) with ever-increasing roles in eukaryotes. Atomic structures and live-cell imaging of transcription, RNA splicing, 3'-end processing, modifications, and degradation machineries provide mechanistic insights, explaining hundreds of diseases caused by their perturbations. This great progress uncovered numerous targets for therapies, some of which have already been successfully exploited, and many opportunities for pharmacological intervention and RNA-guided genome engineering. Myriad unexplained RNAs and RBPs leave the RNA field open for many more exciting discoveries.

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.

Telescripting is a fundamental cotranscriptional gene regulation process that relies on U1 snRNP (U1) to suppress premature 3'-end cleavage and polyadenylation (PCPA) in RNA polymerase II (Pol II) transcripts, which is necessary for full-length transcription of thousands of protein-coding (pre-mRNAs) and long noncoding (lncRNA) genes. Like U1 role in splicing, telescripting requires U1 snRNA base-pairing with nascent transcripts. Inhibition of U1 base-pairing with U1 snRNA antisense morpholino oligonucleotide (U1 AMO) mimics widespread PCPA from cryptic polyadenylation signals (PASs) in human tissues, including PCPA in introns and last exons' 3'-untranslated regions (3' UTRs). U1 telescripting-PCPA balance changes generate diverse RNAs depending on where in a gene it occurs. Long genes are highly U1-telescripting-dependent because of PASs in introns compared to short genes. Enrichment of cell cycle control, differentiation, and developmental functions in long genes, compared to housekeeping and acute cell stress response genes in short genes, reveals a gene size-function relationship in mammalian genomes. This polarization increased in metazoan evolution by previously unexplained intron expansion, suggesting that U1 telescripting could shift global gene expression priorities. We show that that modulating U1 availability can profoundly alter cell phenotype, such as cancer cell migration and invasion, underscoring the critical role of U1 homeostasis and suggesting it as a potential target for therapies. We describe a complex of U1 with cleavage and polyadenylation factors that silences PASs in introns and 3' UTR, which gives insights into U1 telescripting mechanism and transcription elongation regulation.

Genome-wide analyses of the effects of U1 snRNP inhibition in human cells shows that telescripting suppresses premature cleavage and polyadenylation in long introns to sustain expression of large genes important for cell cycle and development. U1 snRNP (U1) functions in splicing introns and telescripting, which suppresses premature cleavage and polyadenylation (PCPA). Using U1 inhibition in human cells, we show that U1 telescripting is selectively required for sustaining long-distance transcription elongation in introns of large genes (median 39 kb). Evidence of widespread PCPA in the same locations in normal tissues reveals that large genes incur natural transcription attrition. Underscoring the importance of U1 telescripting as a gene-size-based mRNA-regulation mechanism, small genes were not sensitive to PCPA, and the spliced-mRNA productivity of ∼1,000 small genes (median 6.8 kb) increased upon U1 inhibition. Notably, these small, upregulated genes were enriched in functions related to acute stimuli and cell-survival response, whereas genes subject to PCPA were enriched in cell-cycle progression and developmental functions. This gene size–function polarization increased in metazoan evolution by enormous intron expansion. We propose that telescripting adds an overarching layer of regulation to size–function-stratified genomes, leveraged by selective intron expansion to rapidly shift gene expression priorities.

Stimulated cells and cancer cells have widespread shortening of mRNA 3’-utranslated regions (3’UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in the last exon and in introns. U1 snRNA (U1), vertebrates’ most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread mRNA shortening. Here we show that U1 AMO also modulates cancer cells’ phenotype, dose-dependently increasing 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 link between U1 regulation and oncogenic and activated cell states, and suggest U1 as a potential target for their modulation.

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., U1 snRNP is a key regulator of mRNA biogenesis through its roles in splicing, and transcription and 3’-end processing. Here the authors show a tumor suppressor-like function of U1 snRNP using in vitro cell migration/invasion assays and transcriptome profiling.

Summary 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.

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.

The survival motor neuron (SMN) protein is the centerfold of a large macromolecular complex comprising SMN, Gemins 2–8, and Unrip. The complex’s best characterized function is in the assembly of a protein ring (Sm core) on small nuclear RNAs (snRNAs), thereby forming small nuclear ribonucleoproteins (snRNPs), the major subunits of cells’ protein-coding mRNA-splicing machine. Biochemical studies identified SMN complex subunits and dissected how this first-of-its-kind RNP assembly device operates an snRNP biogenesis pathway. Atomic structures of a key intermediate provide important mechanistic insights and explain the basis of several spinal muscular atrophy (SMA)-causing SMN mutations. The SMN complex’s interactions with multiple RNA-binding proteins suggest diverse additional roles in RNA metabolism, consistent with SMA’s widespread transcriptome perturbations. Further research on the SMN complex is motivated by its central role in biology and in SMA pathogenesis, aiming to advance prospects of therapy for SMA and potentially other diseases.

Recent observations showed that nascent RNA polymerase II transcripts, pre-mRNAs, and noncoding RNAs are highly susceptible to premature 3′-end cleavage and polyadenylation (PCPA) from numerous intronic cryptic polyadenylation signals (PASs). The importance of this in gene regulation was not previously appreciated as PASs, despite their prevalence, were thought to be active in terminal exons at gene ends. Unexpectedly, antisense oligonucleotide interference with U1 snRNA base-pairing to 5′ splice sites, which is necessary for U1 snRNP’s (U1) function in splicing, caused widespread PCPA in metazoans. This uncovered U1’s PCPA suppression activity, termed telescripting, as crucial for full-length transcription in thousands of vertebrate genes, providing a general role in transcription elongation control. Progressive intron-size expansion in metazoan evolution greatly increased PCPA vulnerability and dependence on U1 telescripting. We describe how these observations unfolded and discuss U1 telescripting’s role in shaping the transcriptome.

Despite intensive research, there are very few reagents with which to modulate and dissect the mRNA splicing pathway. Here, we describe a novel approach to identify such tools, based on detection of the exon junction complex (EJC), a unique molecular signature that splicing leaves on mRNAs. We developed a high-throughput, splicing-dependent EJC immunoprecipitation (EJIPT) assay to quantitate mRNAs spliced from biotin-tagged pre-mRNAs in cell extracts, using antibodies to EJC components Y14 and eukaryotic translation initiation factor 4aIII (eIF4AIII). Deploying EJIPT we performed high-throughput screening (HTS) in conjunction with secondary assays to identify splicing inhibitors. We describe the identification of 1,4-naphthoquinones and 1,4-heterocyclic quinones with known anticancer activity as potent and selective splicing inhibitors. Interestingly, and unlike previously described small molecules, most of which target early steps, our inhibitors represented by the benzothiazole-4,7-dione, BN82685, block the second of two trans-esterification reactions in splicing, preventing the release of intron lariat and ligation of exons. We show that BN82685 inhibits activated spliceosomes' elaborate structural rearrangements that are required for second-step catalysis, allowing definition of spliceosomes stalled in midcatalysis. EJIPT provides a platform for characterization and discovery of splicing and EJC modulators.

Despite equal snRNP stoichiometry in spliceosomes, U1 snRNP (U1) is typically the most abundant vertebrate snRNP. Mechanisms regulating U1 overabundance and snRNP repertoire are unknown. In Sm-core assembly, a key snRNP-biogenesis step mediated by the SMN complex, the snRNA-specific RNA-binding protein (RBP) Gemin5 delivers pre-snRNAs, which join SMN-Gemin2-recruited Sm proteins. We show that the human U1-specific RBP U1-70K can bridge pre-U1 to SMN-Gemin2-Sm, in a Gemin5-independent manner, thus establishing an additional and U1-exclusive Sm core-assembly pathway. U1-70K hijacks SMN-Gemin2-Sm, enhancing Sm-core assembly on U1s and inhibiting that on other snRNAs, thereby promoting U1 overabundance and regulating snRNP repertoire. SMN-Gemin2's ability to facilitate transactions between different RBPs and RNAs explains its multi-RBP valency and the myriad transcriptome perturbations associated with SMN deficiency in neurodegenerative spinal muscular atrophy. We propose that SMN-Gemin2 is a versatile hub for RNP exchange that functions broadly in RNA metabolism.

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 (

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.

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.

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 (∼80%), resulting in a highly unstable and almost undetectable protein (SMNΔ7). We show that this splicing defect creates a potent degradation signal (degron; SMNΔ7-DEG) at SMNΔ7's C-terminal 15 amino acids. The S270A mutation inactivates SMNΔ7-DEG, generating a stable SMNΔ7 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 SMNΔ7-DEG activity.

Gemin5 binds specifically to the small nuclear RNA (snRNA)-defining small nuclear ribonucleoprotein (snRNP) code sequence and is essential, together with other components of the survival of motor neurons (SMN) complex, for the biogenesis of snRNPs, the major constituents of spliceosomes. We show that this binding is mediated by Gemin5's WD repeat domain, a common domain not previously known to bind RNA independently. The entire WD repeat domain, comprising 13 WD motifs, is both necessary and sufficient for sequence-specific, high-affinity binding of Gemin5 to its RNA targets. Using an RNA-mediated hydroxyl radical probing method and mass spectrometry, we mapped a discrete region of the WD repeat domain that contacts snRNAs and demonstrated by mutagenesis that specific amino acids in this region are crucial for Gemin5-snRNA binding. The WD repeat domain is thus a previously undescribed RNA binding domain, and we suggest that the presence of WD repeats should be considered as predictive of potential function in RNA binding.

Cellular functions depend on numerous protein-coding and non-coding 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 mis-regulation 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.

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 (SMA), a common motor neuron degenerative disease of severity commensurate with SMN levels, and correspondingly, snRNP assembly decrease. 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 β-lapachone (in vivo IC50=0.45 μM) 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 a novel ROS target, suggesting that it may play a role in oxidative stress pathophysiology, which is associated with many degenerative diseases.

The survival of motor neurons (SMN) complex is essential for the biogenesis of small nuclear ribonucleoprotein (snRNP) complexes in eukaryotic cells. Reduced levels of SMN cause the motor neuron degenerative disease, spinal muscular atrophy. We identify here stable subunits of the SMN complex that do not contain SMN. Sedimentation and immunoprecipitation experiments using cell extracts reveal at least three complexes composed of Gemin3, -4, and -5; Gemin6, -7, and unrip; and SMN with Gemin2, as well as free Gemin5. Complexes containing Gemin3-Gemin4-Gemin5 and Gemin6-Gemin7-unrip persist at similar levels when SMN is reduced. In cells, immunofluorescence microscopy shows differential localization of Gemin5 after cell stress. We further show that the Gemin5-containing subunits bind small nuclear RNA independently of the SMN complex and without a requirement for exogenous ATP. ATP hydrolysis is, however, required for displacement of small nuclear RNAs from the Gemin5-containing subunits and their assembly into snRNPs. These findings demonstrate a modular nature of the SMN complex and identify a new intermediate in the snRNP assembly process.

Gemin5 is a 170-kDa WD-repeat-containing protein that was initially identified as a component of the survival of motor neurons (SMN) complex. We now show that Gemin5 facilitates the activation of apoptosis signal-regulating kinase 1 (ASK1) and downstream signaling. Gemin5 physically interacted with ASK1 as well as with the downstream kinases SEK1 and c-Jun NH(2)-terminal kinase (JNK1), and it potentiated the H(2)O(2)-induced activation of each of these kinases in intact cells. Moreover, Gemin5 promoted the binding of ASK1 to SEK1 and to JNK1, as well as the ASK1-induced activation of JNK1. In comparison, Gemin5 did not physically associate with MKK7, MKK3, MKK6, or p38. Furthermore, depletion of endogenous Gemin5 by RNA interference (RNAi) revealed that Gemin5 contributes to the activation of ASK1 and JNK1, and to apoptosis induced by H(2)O(2) and tumor necrosis factor-alpha (TNFalpha) in HeLa cells. Together, our results suggest that Gemin5 functions as a scaffold protein for the ASK1-JNK1 signaling module and thereby potentiates ASK1-mediated signaling events.

TDP-43 was recently identified as the major disease protein in neuronal inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). TDP-43 becomes redistributed from the nucleus to the cytoplasm, ubiquitinated, hyperphosphorylated and cleaved to generate C-terminal fragments, thereby linking mismetabolism of TDP-43 to the pathogenesis of FTLD-U. The function of TDP-43 is unclear, however it has been shown that TDP-43 might act as transcription repressor and activator of exon skipping through interaction with proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) family as well as a scaffold for nuclear bodies through interactions with survival motor neuron protein. To investigate whether these binding partners might be associated with TDP-43 pathology, we studied the expression and localization of proteins of the hnRNP family (hnRNP A1, A2/B1, C1/C2) and SMN protein in affected brain regions in patients with sporadic and familial FTLD-U and normal control brains by immunohistochemistry and biochemical analysis. In contrast to TDP-43, no changes in subcellular distribution, no labeling of pathologic inclusions and no biochemical alterations were detectable for the tested hnRNPs and SMN in FTLD-U brains compared to controls. These results argue against a role of these binding partners in the pathogenesis of FTLD-U and emphasize the specificity of TDP-43 as marker for FTLD-U pathology.

On the wonderful occasion of the RNA journal's 20th anniversary, I think one can say without a doubt that the RNA field has been an amazing source of excitement and remarkable advances that have inseminated multiple fields across biology and medicine. The RNA journal reflects the vibrant and collegial RNA community, but the journal's success is also a tribute to the superb leadership, integrity, and scholarship of its Editor, Tim Nilsen, and dedicated Editorial Board. I feel very fortunate to have been working in this exciting field no less because of the many friendships I have made with wonderful colleagues and mostly the truly exceptional individuals that I have had in my laboratory. The journey, so far, has been phenomenal and full of surprises. Looking back, I would not have been able to foresee the course that my laboratory took since the journal's inception. I will briefly describe some highlights that stand out in my mind from our own research and how we got there. I regret that the concise format precludes mentioning the major contributions and influence that others have had on our work—they are many! My laboratory has a long-standing interest in RNA-binding proteins, RNA-protein complexes (RNPs), and their roles in gene regulation and disease. Most gene regulation in complex eukaryotes occurs post-transcriptionally and is mediated by RNA-binding proteins and small noncoding RNAs. To produce mRNA, the primary gene transcripts of the majority of protein coding genes (pre-mRNAs; historically hnRNAs) are extensively processed to remove translation open reading frame-disrupting introns by splicing, and by 5′-end capping and 3′-end cleavage and polyadenylation (CPA). Splicing is mediated by the spliceosome, comprised of non-coding small nuclear RNPs (major: U1, U2, U4, U5, U6; minor: U11, U12, U4atac, U6atac) and protein factors. The majority of pre-mRNAs have multiple introns, each with 5′- and 3′-splice sites (ss) and multiple polyadenylation signals (PASs) that can be utilized in various alternative combinations to produce diverse mRNA and protein isoforms from the same gene. Pre-mRNA processing initiates co-transcriptionally and completes in the nucleus. The mRNAs are then transported to the cytoplasm where they can be translated and are subsequently degraded. Each of these events is regulated in a cell type and cell cycle stage-dependent manner and in response to external cues. All these processes, including recognition of constitutive and alternative pre-mRNA processing signals, depend on RNA-binding proteins (RBPs or RNP proteins).

The survival of motor neurons protein (SMN) is part of a large complex that contains six other proteins, Gemins2-7. The SMN complex assembles the heptameric Sm protein core on small nuclear RNAs (snRNAs) and plays a critical role in the biogenesis of snRNPs, the major and essential components of mRNA splicing in eukaryotes. For its function, the SMN complex binds Sm proteins and snRNAs, which it distinguishes from other RNAs by specific features they contain. We show here that Gemin5, a 170 kDa WD-repeat protein, is the snRNA binding protein of the SMN complex. Gemin5 binds directly and specifically to the unique features, including the Sm site, of snRNAs. Reduction of Gemin5 results in reduced capacity of the SMN complex to bind snRNAs and to assemble Sm cores. Gemin5 therefore functions as the factor that allows the SMN complex to distinguish snRNAs from other cellular RNAs for snRNP biogenesis.

In eukaryotic cells, the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and likely other RNPs is mediated by an assemblyosome, the survival of motor neurons (SMN) complex. The SMN complex, composed of SMN and the Gemins (2-7), binds to the Sm proteins and to snRNAs and constructs the heptameric rings, the common cores of Sm proteins, on the Sm site (AU(56)G) of the snRNAs. We have determined the specific sequence and structural features of snRNAs for binding to the SMN complex and Sm core assembly. The minimal SMN complex-binding domain in snRNAs (except U1) is composed of an Sm site and a closely adjacent 3'stem-loop. Remarkably, the specific sequence of the stemloop is not important for SMN complex binding, but it must be located within a short distance of the 3'end of the RNA for an Sm core to assemble. This minimal snRNA-defining "snRNP code" is recognized by the SMN complex, which binds to it directly and with high affinity and assembles the Sm core. The recognition of the snRNAs is provided by Gemin5, a component of the SMN complex that directly binds the snRNP code. Gemin5 is a novel RNA-binding protein that is critical for snRNP biogenesis. Thus, the SMN complex is the identifier, as well as assembler, of the abundant class of snRNAs in cells. The function of the SMN complex, previously unanticipated because RNP biogenesis was believed to occur by self-assembly, confers stringent specificity on otherwise potentially illicit RNA-protein interactions.

Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN, together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels, as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known. To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts. Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a biochemical activity in cells from patients with spinal muscular atrophy.

Y14 is a component of the splicing-dependent exon-exon junction complex (EJC) and is involved in the mRNA quality control system called nonsense-mediated mRNA decay. It has recently been shown that together with another EJC component, Mago, the Drosophila homologue DmY14/Tsunagi is required for proper localization of oskar mRNA during oogenesis, a process critical for posterior formation in Drosophila development. Here we show that the nematode Caenorhabditis elegans Ce-Y14 and MAG-1 (Mago homologue) are required for late embryogenesis and proper germline sexual differentiation. Like in other organisms, Ce-Y14 preferentially binds to spliced mRNA and specifically interacts with MAG-1. Consistent with the evolutionarily conserved interaction between Y14 and Mago homologues, suppression of Ce-Y14 by RNAi resulted in the same phenotypes as those caused by RNAi of mag-1 lethality during late embryogenesis and masculinization of the adult hermaphrodite germline. Our results demonstrate that the evolutionarily conserved interaction between two EJC components, Ce-Y14 and MAG-1, has critical developmental roles in C. elegans.

Dimethylation at arginine residues has been shown to be central in cellular processes such as signal transduction, transcription activation, and protein sorting. The two methyl groups are either placed symmetric or asymmetric on the zeta standing nitrogen atoms of the arginine side chain. Here, we introduce a novel method that enables the localization of dimethylarginine (DMA) residues in gel-separated proteins at a level of sensitivity of better than 1 pmol and that allows one to distinguish between the isomeric symmetric and asymmetric position of the methyl groups. The method utilizes two side-chain fragments of DMA, the dimethylammonium ion (m/z 46.06) and the dimethylcarbodiimidium ion (m/z 71.06), for positive ion mode precursor ion scanning. Dimethylcarbodiimidium ions (m/z 71.06) are produced by symmetric as well as asymmetric dimethylarginine but are observed more strongly for symmetric DMA. It is utilized here in the precursor of m/z 71 scan to indicate the presence of DMA in a peptide. The dimethylammonium ion (m/z 46.06) is specific for asymmetric DMA and is utilized here in the precursor of m/z 46 scan. The positive ion mode allows for the identification of the protein by peptide sequencing and simultaneous detection and localization of the modified residues. The analysis can be conducted on any mass spectrometer capable of precursor ion scanning. However, the high resolution of a quadrupole TOF instrument is beneficial to assign the accurate charge state of the often highly charged precursors. Using the precursor of m/z 71 scan, we found FUS/TLS and Sam68 to be DMA-containing proteins. We discovered at least 20 DMA sites in FUS/TLS. In MS/MS, we observed neutral loss of dimethylamine (m/z 45.05) from which it follows that the dimethylation in FUS/TLS is asymmetric. Monitoring in parallel the fragments m/z 46.06 and 71.06 in precursor ion scans and peptide sequencing, we identified at least nine asymmetric DMA modifications in Sam68. The parallel monitoring of fragments in precursor ion scans is a versatile tool to specify the nature of protein modifications in cases where a single fragment is not conclusive.

International audience; 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.

The survival of motor neurons (SMN) protein is the product of the gene mutated or deleted in the neurodegenerative disease, spinal muscular atrophy. SMN is part of a large macromolecular complex that also contains Gemin2, Gemin3, Gemin4, Gemin5, and Gemin6. The SMN complex functions in the assembly of spliceosomal small nuclear ribonucleoproteins and probably other ribonucleoprotein particles. We have identified a novel protein component of the SMN complex termed Gemin7 using native purified SMN complexes and peptide sequencing by mass spectrometry. Coimmunoprecipitation and immunolocalization experiments demonstrate that Gemin7 is a component of the SMN complex and colocalizes with SMN in the cytoplasm and in gems. Binding experiments show that Gemin7 interacts directly with SMN and Gemin6 and mediates the association of Gemin6 with the SMN complex. The amino acid sequence of Gemin7 does not contain any recognizable motifs with the exception of several arginine and glycine repeats that are necessary for its interaction with SMN. Moreover, Gemin7 interacts with several Sm proteins of spliceosomal small nuclear ribonucleoproteins, in particular, with SmE. With the identification of Gemin7, the inventory of the core components of the SMN complex appears essentially complete.

Background: Y14 is an RNA binding protein which is part of a multiprotein complex, the exon-exon junction complex (EJC), that assembles on the exon-exon junctions of mRNAs produced by splicing. The position-specific binding of Y14 persists on mRNAs after their export to the cytoplasm. Thus, Y14, together with its interacting proteins, has the capacity to communicate to the cytoplasm the processing history of the mRNA, including the position of the removed introns, information that is likely to be important for defining premature termination codons. How Y14 and other components of the EJC are removed from mRNAs into the cytoplasm has not been determined. Results: We show that Y14 but not another EJC component, Aly/REF, is present in polysome profile fractions containing one ribosome per mRNA. Using reporter constructs in an in vitro splicing/translation-coupled system, we show that Y14 remains associated with untranslated mRNAs but is removed from translationally active mRNAs. Importantly, mRNAs whose translation in vivo is prevented by the presence of strong secondary 5′ UTR structure retain Y14 in the cytoplasm. Conclusions: These findings indicate that Y14 remains associated with mRNAs in the cytoplasm until they are translated, and translation is required to remove Y14 from mRNAs. Thus, the process of translation removes the splicing-dependent EJC protein imprints, which most likely function in the surveillance of mRNAs to define premature termination codons and possibly also in modulating the translation activity of cytoplasmic mRNAs.

Gemin3 is a DEAD-box RNA helicase that binds to the Survival of Motor Neurons (SMN) protein and is a component of the SMN complex, which also comprises SMN, Gemin2, Gemin4, Gemin5, and Gemin6. Reduction in SMN protein results in Spinal muscular atrophy (SMA), a common neurodegenerative disease. The SMN complex has critical functions in the assembly/restructuring of diverse ribonucleoprotein (RNP) complexes. Here we report that Gemin3 and Gemin4 are also in a separate complex that contains eIF2C2, a member of the Argonaute protein family. This novel complex is a large ∼15S RNP that contains numerous microRNAs (miRNAs). We describe 40 miRNAs, a few of which are identical to recently described human miRNAs, a class of small endogenous RNAs. The genomic sequences predict that miRNAs are likely to be derived from larger precursors that have the capacity to form stem–loop structures.

From sites of transcription in the nucleus to the outreaches of the cytoplasm, messenger RNAs are associated with RNA-binding proteins. These proteins influence pre-mRNA processing as well as the transport, localization, translation and stability of mRNAs. Recent discoveries have shown that one group of these proteins marks exon exon junctions and has a role in mRNA export. These proteins communicate crucial information to the translation machinery for the surveillance of nonsense mutations and for mRNA localization and translation.

The Survival of Motor Neurons (SMN) is the disease gene of spinal muscular atrophy. We have previously established a genetic system based on the chicken pre-B cell line DT40, in which expression of SMN protein is regulated by tetracycline, to study the function of SMN in vivo. Depletion of SMN protein is lethal to these cells. Here we tested the functionality of mutant SMN proteins by determining their capacity to rescue the cells after depletion of wild-type SMN. Surprisingly, all of the spinal muscular atrophy-associated missense mutations tested were able to support cell viability and proliferation. Deletion of the amino acids encoded by exon 7 of the SMN gene resulted in a partial loss of function. A mutant SMN protein lacking both the tyrosine/glycine repeat (in exon 6) and exon 7 failed to sustain viability, indicating that the C terminus of the protein is critical for SMN activity. Interestingly, the Tudor domain of SMN, encoded by exon 3, does not appear to be essential for SMN function since a mutant deleted of this domain restored cell viability. Unexpectedly, a chicken SMN mutant (DeltaN39) lacking the N-terminal 39 amino acids that encompass the Gemin2-binding domain also rescued the lethal phenotype. Moreover, the level of Gemin2 in DeltaN39-rescued cells was significantly reduced, indicating that Gemin2 is not required for DeltaN39 to perform the essential function of SMN in DT40 cells. These findings suggest that SMN may perform a novel function in DT40 cells.

Background: The survival of motor neurons (SMN) protein is the protein product of the spinal muscular atrophy (SMA) disease gene. SMN and its associated proteins Gemin2, Gemin3, and Gemin4 form a large complex that plays a role in snRNP assembly, pre-mRNA splicing, and transcription. The functions of SMN in these processes are mediated by a direct interaction of SMN with components of these machineries, such as Sm proteins and RNA helicase A. Results: We show that SMN binds directly to fibrillarin and GAR1. Fibrillarin and GAR1 are specific markers of the two classes of small nucleolar ribonucleoprotein particles (snoRNPs) that are involved in posttranscriptional processing and modification of ribosomal RNA. SMN interaction requires the arginine- and glycine-rich domains of both fibrillarin and GAR1 and is defective in SMN mutants found in some SMA patients. Coimmunoprecipitations demonstrate that the SMN complex associates with fibrillarin and with GAR1 in vivo. The inhibition of RNA polymerase I transcription causes a transient redistribution of SMN to the nucleolar periphery and loss of fibrillarin and GAR1 colocalization with SMN in gems. Furthermore, the expression of a dominant-negative mutant of SMN (SMNΔN27) causes snoRNPs to accumulate outside of the nucleolus in structures that also contain components of gems and coiled (Cajal) bodies. Conclusions: These findings identify fibrillarin and GAR1 as novel interactors of SMN and suggest a function for the SMN complex in the assembly and metabolism of snoRNPs. We propose that the SMN complex performs functions necessary for the biogenesis and function of diverse ribonucleoprotein complexes.

The survival of motor neurons protein (SMN), the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, functions as an assembly factor for snRNPs and likely other RNPs. SMN binds the arginine- and glycine-rich (RG) domains of the snRNP proteins SmD1 and SmD3. Specific arginines in these domains are modified to dimethylarginines, a common modification of unknown function. We show that SMN binds preferentially to the dimethylarginine-modified RG domains of SmD1 and SmD3. The binding of other SMN-interacting proteins is also strongly enhanced by methylation. Thus, methylation of arginines is a novel mechanism to promote specific protein-protein interactions and appears to be key to generating high-affinity SMN substrates. It is reasonable to expect that protein hypomethylation may contribute to the severity of SMA.

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.

The motor neuron degenerative disease spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. Here we report a genetic system developed in the chicken pre-B cell line DT40, in which the endogenous SMN gene is disrupted by homologous recombination, and SMN protein is expressed from a chicken SMN cDNA under control of a tetracycline (tet)-repressible promoter. Addition of tet results in depletion of SMN protein and consequent cell death, which directly demonstrates that SMN is required for cell viability. The tet-induced lethality can be rescued by expression of human SMN, indicating that the function of SMN is highly conserved between the two species. Cells expressing low levels of SMN display slow growth proportional to the amount of SMN they contain. Interestingly, the level of the SMN-interacting protein Gemin2 decreases significantly following depletion of SMN, supporting the conclusion that SMN and Gemin2 form a stable complex in vivo. This system provides a powerful setting for studying the function of SMN in vivo and for screening for potential therapeutics for spinal muscular atrophy.

Immunohistochemical techniques were employed to examine the distribution of RNA-binding proteins A2 and B1 in the rat forebrain. Intense A2 and B1 immunolabeling were observed in the nucleoplasm of the neurons in the cerebral cortices, hippocampal formation, olfactory regions, caudate-putamen as well as the supraoptic nucleus of hypothalamus. In contrast, within the bed nucleus of the stria terminalis, as well as the medial and lateral habenular nucleus of thalamus, immunoreactivity for both proteins was weak. Within the globus pallidus and thalamic nucleus immunoreactivity for A2 was hardly detectable despite of intense B1 immunolabeling, while within the endopiriform nucleus and lateral and basolateral nucleus of amygdala intensity of B1 immunolabeling was relatively weak compared to A2. Our study suggests that the distribution of A2 and B1 are not constant throughout the forebrain and this diversity may reflect the post-transcriptional regulation of cellspecific gene expression of neuronal cells. NeuroReport 11:3099‐3102 & 2000 Lippincott Williams & Wilkins.

Spinal muscular atrophy is a common often lethal neurodegenerative disease resulting from deletions or mutations in the survival motor neuron gene (SMN). SMN is ubiquitously expressed in metazoan cells and plays a role in small nuclear ribonucleoprotein assembly and pre-mRNA splicing. Here we characterize the Schizosacharomyces pombe orthologue of SMN (yeast SMN (ySMN)). We report that the ySMN protein is essential for viability and localizes in both the cytoplasm and the nucleus. Like human SMN, we show that ySMN can oligomerize. Remarkably, ySMN interacts directly with human SMN and Sm proteins. The highly conserved carboxyl-terminal domain of ySMN is necessary for the evolutionarily conserved interactions of SMN and required for cell viability. We also demonstrate that the conserved amino-terminal region of ySMN is not required for SMN and Sm binding but is critical for the housekeeping function of SMN.

The survival of motor neurons (SMN) protein, the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, is localized both in the cytoplasm and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins including Gemin2 (formerly SIP1) and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with snRNP Sm core proteins and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing by serving in the regeneration of spliceosomes. These functions are likely impaired in cells of SMA patients because they have reduced levels of functional SMN. Here, we report the identification by nanoelectrospray mass spectrometry of a novel component of the SMN complex that we name Gemin4. Gemin4 is associated in vivo with the SMN complex through a direct interaction with Gemin3. The tight interaction of Gemin4 with Gemin3 suggests that it could serve as a cofactor of this DEAD box protein. Gemin4 also interacts directly with several of the Sm core proteins. Monoclonal antibodies against Gemin4 efficiently immunoprecipitate the spliceosomal U snRNAs U1 and U5 from Xenopus oocytes cytoplasm. Immunolocalization experiments show that Gemin4 is colocalized with SMN in the cytoplasm and in gems. Interestingly, Gemin4 is also detected in the nucleoli, suggesting that the SMN complex may also function in preribosomal RNA processing or ribosome assembly.

The human immunodeficiency virus type-1 Rev protein induces the nuclear export of intron-containing viral mRNAs that harbor its binding site, the Rev response element (RRE). A leucine-rich region of Rev, the activation domain, is essential for function and has been shown to be a nuclear export signal (NES). Although Rev exports viral RNAs that resemble cellular mRNAs, competition studies performed using microinjected Xenopus laevis oocytes have previously indicated that Rev utilizes a non-mRNA export pathway. Here, we show that Rev is able to induce the export of both spliceable and non-spliceable RRE-containing pre-mRNAs and that this activity is not dependent on the location of the RRE within the RNA. Importantly, even RNA molecules of different classes, such as U3 snoRNA and U6 snRNA, which are retained in the nucleus by non-pre-mRNA mechanisms, are exported to the cytoplasm in response to Rev. Consistent with the notion that Rev-mediated export of RRE-containing RNA is mechanistically distinct from the export of processed cellular mRNA, a chimeric Rev protein in which its NES is replaced by the NES of hnRNP A1 does not induce the export of a Rev-responsive mRNA. Finally, we demonstrate that Rev/RRE-activated RNA export is, like other nuclear export pathways, linked to the Ran-GTPase cycle.

Spinal muscular atrophy (SMA) is a common motor neuron degenerative disease and the leading genetic cause of death of young children. The survival of motor neurons ( SMN ) gene, the SMA disease gene, is homozygously deleted or mutated in more than 98% of SMA patients. The SMN protein interacts with itself, with SMN-interacting protein 1, and with several spliceosomal small nuclear ribonucleoprotein (snRNP) Sm proteins. A complex containing SMN plays a critical role in spliceosomal snRNP assembly and in pre-mRNA splicing. SMN mutants found in SMA patients show reduced self-association and lack the capacity to regenerate the splicing machinery. Here we demonstrate that SMN mutants found in SMA patients are defective in binding to Sm proteins. Moreover, we show that SMN, but not mutants found in SMA patients, can form large oligomers and that SMN oligomerization is required for high-affinity binding to spliceosomal snRNP Sm proteins. These findings directly link the impaired interaction between SMN and Sm proteins to a defect in snRNP metabolism and to SMA.

We employed a phage display system to search for proteins that interact with transportin 1 (TRN1), the import receptor for shuttling hnRNP proteins with an M9 nuclear localization sequence (NLS), and identified a short region within the N-terminus of the nucleoporin Nup153 which binds TRN1. Nup153 is located at the nucleoplasmic face of the nuclear pore complex (NPC), in the distal basket structure, and functions in mRNA export. We show that this Nup153 TRN1-interacting region is an M9 NLS. We found that both import and export receptors interact with several regions of Nup153, in a RanGTP-regulated fashion. RanGTP dissociates Nup153-import receptor complexes, but is required for Nup153-export receptor interactions. We also show that Nup153 is a RanGDP-binding protein, and that the interaction is mediated by the zinc finger region of Nup153. This represents a novel Ran-binding domain, which we term the zinc finger Ran-binding motif. We provide evidence that Nup153 shuttles between the nuclear and cytoplasmic faces of the NPC. The presence of an M9 shuttling domain in Nup153, together with its ability to move within the NPC and to interact with export receptors, suggests that this nucleoporin is a mobile component of the pore which carries export cargos towards the cytoplasm.

hnRNP A2/B1 proteins are among the most abundant pre-mRNA-binding proteins of vertebrates and structurally similar to hnRNP A1. We have produced two specific monoclonal antibodies against A2 and B1 and studied their molecular characteristics and in vivo expression in rat tissues. Immunoprecipitation demonstrated that the hnRNP A2/B1 complexes contain many snRNP (small nuclear ribonucleoprotein) proteins, consistent with their role in pre-mRNA splicing. RNA polymerase II inhibition causes nucleocytoplasmic shuttling of A2 and B1. In most tissues, they are localized in the nucleus; however, in the squamous epithelium of the skin and esophagus A2 is also distributed in the cytoplasm. The relative amounts of A2 and B1 are not constant among different tissues. In the adrenal, only A2 is extremely abundant in the medulla but not in the cortex. In the testis the expression of A2 and B1 are observed through spermatogenesis, and different from A1 which is stringently repressed in spermatocytes. We also found and cloned a novel testis-specific isoform of A2/B1, namely hnRNP B0. The difference of expression of A2, B1, and A1 provides new information on their in vivo roles. The diversity of A/B group hnRNP proteins may have important effects on the posttranscriptional regulation of cell-specific gene expression. © 1999 Academic Press

The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.