Sm Proteins Research Articles

Arginine methylation controls the strength of γc-family cytokine signaling in T cell maintenance.

The methylation of arginine residues in proteins is a post-translational modification that contributes to a wide range of biological processes. Many cytokines involved in T cell development and activation utilize the common cytokine receptor γ-chain (γc) and the kinase JAK3 for signal transduction, but the regulatory mechanism that underlies the expression of these factors remains unclear. Here we found that the arginine methyltransferase PRMT5 was essential for the maintenance of invariant natural killer T cells (iNKT cells), CD4+ T cells and CD8+ T cells. T cell-specific deletion of Prmt5 led to a marked reduction in signaling via γc-family cytokines and a substantial loss of thymic iNKT cells, as well as a decreased number of peripheral CD4+ T cells and CD8+ T cells. PRMT5 induced the symmetric dimethylation of Sm proteins that promoted the splicing of pre-mRNA encoding γc and JAK3, and this critically contributed to the expression of γc and JAK3. Thus, arginine methylation regulates strength of signaling via γc-family cytokines by facilitating the expression of signal-transducing components.

PRMT5 inhibition promotes osteogenic differentiation of mesenchymal stromal cells and represses basal interferon stimulated gene expression.

Protein arginine methyltransferases (PRMTs) catalyze symmetric and asymmetric methylation on arginine residues of multiple protein targets including histones and have essential roles in organismal development and disease. PRMT5 mediates symmetric di-methylation (sDMA) of arginine 2 (H3R2me2s) and arginine 8 on histone 3 (H3R8me2s), arginine 3 on histones 2A and 4 (H2A/H4R3me2s) as well as several non-histone substrates like Sm proteins. Here, we found that selective inhibition of PRMT5 in mesenchymal stromal cells (MSCs) led to a reduction in colony forming units (CFUs) and increased osteoblast differentiation. PRMT5 inhibition blocked global symmetric dimethylation of H3R8 and H4R3 but not on H3R2. Genome-wide expression analysis by total RNA sequencing of mesenchymal stromal cells undergoing osteogenic differentiation revealed significant reduction in the intrinsic expression of several interferon-stimulated genes (ISGs) upon PRMT5 inhibition. Effects of PRMT5 inhibition on basal ISG expression and osteogenic differentiation was effectively blocked by exogenous activation of type I IFN signaling. Together, these results indicate important functions for PRMT5 in the regulation of basal interferon gene expression in MSCs and in the control of differentiation potential of MSCs during osteogenic differentiation.

Comparison of the uridine-binding site of hexameric and heptameric archaeal Lsm proteins.

Comparison of the uridine-binding site of hexameric and heptameric archaeal Lsm proteins.

A genome-wide screen of Epstein-Barr virus proteins that modulate host SUMOylation identifies a SUMO E3 ligase conserved in herpesviruses.

Many cellular processes pertinent for viral infection are regulated by the addition of small ubiquitin-like modifiers (SUMO) to key regulatory proteins, making SUMOylation an important mechanism by which viruses can commandeer cellular pathways. Epstein-Barr virus (EBV) is a master at manipulating of cellular processes, which enables life-long infection but can also lead to the induction of a variety of EBV-associated cancers. To identify new mechanisms by which EBV proteins alter cells, we screened a library of 51 EBV proteins for global effects on cellular SUMO1 and SUMO2 modifications (SUMOylation), identifying several proteins not previously known to manipulate this pathway. One EBV protein (BRLF1) globally induced the loss of SUMOylated proteins, in a proteasome-dependent manner, as well as the loss of promeylocytic leukemia nuclear bodies. However, unlike its homologue (Rta) in Kaposi’s sarcoma associated herpesvirus, it did not appear to have ubiquitin ligase activity. In addition we identified the EBV SM protein as globally upregulating SUMOylation and showed that this activity was conserved in its homologues in herpes simplex virus 1 (HSV1 UL54/ICP27) and cytomegalovirus (CMV UL69). All three viral homologues were shown to bind SUMO and Ubc9 and to have E3 SUMO ligase activity in a purified system. These are the first SUMO E3 ligases discovered for EBV, HSV1 and CMV. Interestingly the homologues had different specificities for SUMO1 and SUMO2, with SM and UL69 preferentially binding SUMO1 and inducing SUMO1 modifications, and UL54 preferentially binding SUMO2 and inducing SUMO2 modifications. The results provide new insights into the function of this family of conserved herpesvirus proteins, and the conservation of this SUMO E3 ligase activity across diverse herpesviruses suggests the importance of this activity for herpesvirus infections.

Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology.

ABSTRACTSpinal muscular atrophy (SMA) is an inherited neurodegenerative condition caused by a reduction in the amount of functional survival motor neuron (SMN) protein. SMN has been implicated in transport of mRNA in neural cells for local translation. We previously identified microtubule-dependent mobile vesicles rich in SMN and SNRPB, a member of the Sm family of small nuclear ribonucleoprotein (snRNP)-associated proteins, in neural cells. By comparing the interactomes of SNRPB and SNRPN, a neural-specific Sm protein, we now show that the essential neural protein neurochondrin (NCDN) interacts with Sm proteins and SMN in the context of mobile vesicles in neurites. NCDN has roles in protein localisation in neural cells and in maintenance of cell polarity. NCDN is required for the correct localisation of SMN, suggesting they may both be required for formation and transport of trafficking vesicles. NCDN may have potential as a therapeutic target for SMA together with, or in place of the targeting of SMN expression.This article has an associated First Person interview with the first author of the paper.

Signature microRNAs of nuclear Sm complex associated with breast cancer tumorigenesis.

It is well known that Sm proteins, small nuclear ribonucleoproteins, act as core spliceosomal factors in alternative splicing of mRNA precursors. MicroRNAs (miRNAs) can function in alternative splicing by targeting mRNAs of splicing factors. However, the direct interaction between miRNAs and proteins of splicing complex in nucleus has not been explored. In this study, the mature miRNAs in nuclear Sm complex of breast cancer cells and normal breast epithelial cells were characterized. Small RNA sequencing of immunoprecipitated nuclear Sm complex with the SmD1-specific antibody identified 123 and 170 mature miRNAs in nuclear Sm complex of normal breast cells and breast cancer cells, respectively. The results of Northern blot analysis confirmed the existence of mature miRNAs in Sm complex and electrophoretic mobility shift assay (EMSA) validated the binding of miRNAs with proteins of Sm complex. Among the identified miRNAs bound to the Sm complex in nucleus, 94 miRNAs were significantly upregulated, and 39 miRNAs significantly downregulated in breast cancer cells compared with normal breast cells, suggesting that miRNAs in nuclear Sm complex might be associated to tumorigenesis of breast cancer by regulating Sm complex during alternative splicing of mRNA precursors. Our study provided novel clues to reveal the regulatory mechanism of Sm complex in the assembly of spliceosome and contributed novel aspects of miRNAs to tumorigenesis of breast cancer.

The Sm-core mediates the retention of partially-assembled spliceosomal snRNPs in Cajal bodies until their full maturation.

Cajal bodies (CBs) are nuclear non-membrane bound organelles where small nuclear ribonucleoprotein particles (snRNPs) undergo their final maturation and quality control before they are released to the nucleoplasm. However, the molecular mechanism how immature snRNPs are targeted and retained in CBs has yet to be described. Here, we microinjected and expressed various snRNA deletion mutants as well as chimeric 7SK, Alu or bacterial SRP non-coding RNAs and provide evidence that Sm and SMN binding sites are necessary and sufficient for CB localization of snRNAs. We further show that Sm proteins, and specifically their GR-rich domains, are important for accumulating snRNPs in CBs. Accordingly, core snRNPs containing the Sm proteins, but not naked snRNAs, restore the formation of CBs after their depletion. Finally, we show that immature but not fully assembled snRNPs are able to induce CB formation and that microinjection of an excess of U2 snRNP-specific proteins, which promotes U2 snRNP maturation, chases U2 snRNA from CBs. We propose that the accessibility of the Sm ring represents the molecular basis for the quality control of the final maturation of snRNPs and the sequestration of immature particles in CBs.

Producing Hfq/Sm Proteins and sRNAs for Structural and Biophysical Studies of Ribonucleoprotein Assembly.

Hfq is a bacterial RNA-binding protein that plays key roles in the post-transcriptional regulation of gene expression. Like other Sm proteins, Hfq assembles into toroidal discs that bind RNAs with varying affinities and degrees of sequence specificity. By simultaneously binding to a regulatory small RNA (sRNA) and an mRNA target, Hfq hexamers facilitate productive RNA∙∙∙RNA interactions; the generic nature of this chaperone-like functionality makes Hfq a hub in many sRNA-based regulatory networks. That Hfq is crucial in diverse cellular pathways-including stress response, quorum sensing, and biofilm formation-has motivated genetic and "RNAomic" studies of its function and physiology (in vivo), as well as biochemical and structural analyses of Hfq∙∙∙RNA interactions (in vitro). Indeed, crystallographic and biophysical studies first established Hfq as a member of the phylogenetically conserved Sm superfamily. Crystallography and other biophysical methodologies enable the RNA-binding properties of Hfq to be elucidated in atomic detail, but such approaches have stringent sample requirements, viz.: reconstituting and characterizing an Hfq·RNA complex requires ample quantities of well-behaved (sufficient purity, homogeneity) specimens of Hfq and RNA (sRNA, mRNA fragments, short oligoribonucleotides, or even single nucleotides). The production of such materials is covered in this chapter, with a particular focus on recombinant Hfq proteins for crystallization experiments.

Truncated forms of U2 snRNA (U2-tfs) are shunted toward a novel uridylylation pathway that differs from the degradation pathway for U1-tfs

ABSTRACTDuring the biogenesis of U1 small nuclear ribonucleoprotein, a small population of U1 snRNA molecules acquires an extra methylation at the first transcribed nucleotide and a nucleolytic cleavage to remove the 3′ structured region including the Sm protein–binding site and stem-loop 4. These modifications occur before hypermethylation of the monomethylated 5′ cap, whereby producing truncated forms of U1 snRNA (U1-tfs) that are diverted from the normal pathway to a processing body–associated degradation pathway. Here, we demonstrate that a small population of U2 snRNA molecules receives post-transcriptional modifications similar to those of U1 to yield U2-tfs. Like U1-tfs, U2-tfs molecules were produced from transcripts of the U2 snRNA gene having all cis-elements or lacking the 3′ box. Unlike U1-tfs, however, a portion of U2-tfs received additional uridylylation of up to 5 nucleotides in length at position 87 (designated as U2-tfs-polyU) and formed an Sm protein–binding site–like structure that was stabilized by the small nuclear ribonucleoprotein SmB/B' probably as a part of heptameric Sm core complex that associates to the RNA. Both U2-tfs and U2-tfs-polyU were degraded by a nuclease distinct from the canonical Dis3L2 by a process catalyzed by terminal uridylyltransferase 7. Collectively, our data suggest that U2 snRNA biogenesis is regulated, at least in part, by a novel degradation pathway to ensure that defective U2 molecules are not incorporated into the spliceosome.

A viral Sm-class RNA base-pairs with mRNAs and recruits microRNAs to inhibit apoptosis.

Viruses express several classes of non-coding RNAs; the functions and mechanisms by which most of these act are unknown. Herpesvirus saimiri, a γ-herpesvirus that establishes latency in the T cells of New World primates and has the ability to cause aggressive leukaemias and lymphomas in non-natural hosts, expresses seven small nuclear uracil-rich non-coding RNAs (called HSURs) in latently infected cells. These HSURs associate with Sm proteins, and share biogenesis and structural features with cellular Sm-class small nuclear RNAs. One of these HSURs (HSUR2) base-pairs with two host cellular microRNAs (miR-142-3p and miR-16) but does not affect their abundance or activity, which suggests that its interactions with them perform alternative functions. Here we show that HSUR2 also base-pairs with mRNAs in infected cells. We combined in vivo psoralen-mediated RNA-RNA crosslinking and high-throughput sequencing to identify the mRNAs targeted by HSUR2, which include mRNAs that encode retinoblastoma and factors involved in p53 signalling and apoptosis. We show that HSUR2 represses the expression of target mRNAs and that base-pairing between HSUR2 and miR-142-3p and miR-16 is essential for this repression, suggesting that HSUR2 recruits these two cellular microRNAs to its target mRNAs. Furthermore, we show that HSUR2 uses this mechanism to inhibit apoptosis. Our results uncover a role for this viral Sm-class RNA as a microRNA adaptor in the regulation of gene expression that follows precursor mRNA processing.

Physico-Chemical Properties of Soybean Meal-Based Adhesives Reinforced by Ethylene Glycol Diglycidyl Ether and Modified Nanocrystalline Cellulose.

An eco-friendly soybean meal-based adhesive (SM adhesive) was developed by incorporating ethylene glycol diglycidyl ether (EGDE) and nanocrystalline cellulose (NCC). In order to introduce epoxy groups, NCC was modified by KH560 (denoted as MNCC). The functional groups, thermal stability, and cross section of the resultant adhesive were characterized. Three-ply plywood was fabricated to measure the dry and wet shear strength of the adhesive. The experimental results showed that the epoxy groups on MNCC reacted with the carboxyl group of SM protein molecules, forming a crosslinking network and a ductile adhesive layer. As a result, compared with the SM adhesive modified by EGDE, the thermal stability of the adhesive with MNCC was improved and the wet shear strength was increased to 1.08 MPa.

High glucose downregulates myocardin expression in rat glomerular mesangial cells via the ERK signaling pathway.

Mesangial cells (MCs), which are vascular smooth muscle-derived cells, occupy the central position in the glomerulus. Diabetic nephropathy (DN) is one of the most common diabetes complications and is likely attributed to the loss of MC contractility. Myocardin stimulates downstream vascular smooth muscle genes and regulates the contractility of vascular smooth muscle cells. Therefore, we hypothesized that myocardin is expressed in MCs and that high glucose is involved in the regulation of myocardin and downstream contractile genes in the context of DN. Confocal microscopy revealed that myocardin is expressed in rat MCs. Western blot and RT-qPCR analyses showed that treatment with 30 mM D-glucose significantly downregulated the mRNA and protein levels of myocardin and downstream SM α-actin. As an isotonic contrast, 30 mM mannitol did not affect myocardin mRNA levels but did downregulate myocardin protein levels. Treatment with 30 mM mannitol also downregulated SM α-actin mRNA and protein levels. Conversely, as another isotonic contrast, 30 mM L-glucose also had no effect on myocardin and SM α-actin expression in MCs. The extracellular signal-regulated kinase (ERK) pathway was activated by treatment with 30 mM D-glucose or mannitol, while specific inhibitors of the ERK pathway (PD98059) compromised the downregulation of myocardin and SM α-actin triggered by high glucose or mannitol. Thus we revealed that myocardin is expressed in MCs and that high glucose downregulates myocardin expression and downstream contractile protein SM α-actin via the ERK pathway. Our results suggest a novel mechanism for high glucose inhibition of MC contraction, which contributes to DN pathogenesis.

Acidic C-terminal domains autoregulate the RNA chaperone Hfq.

The RNA chaperone Hfq is an Sm protein that facilitates base pairing between bacterial small RNAs (sRNAs) and mRNAs involved in stress response and pathogenesis. Hfq possesses an intrinsically disordered C-terminal domain (CTD) that may tune the function of the Sm domain in different organisms. In Escherichia coli, the Hfq CTD increases kinetic competition between sRNAs and recycles Hfq from the sRNA-mRNA duplex. Here, de novo Rosetta modeling and competitive binding experiments show that the acidic tip of the E. coli Hfq CTD transiently binds the basic Sm core residues necessary for RNA annealing. The CTD tip competes against non-specific RNA binding, facilitates dsRNA release, and prevents indiscriminate DNA aggregation, suggesting that this acidic peptide mimics nucleic acid to auto-regulate RNA binding to the Sm ring. The mechanism of CTD auto-inhibition predicts the chaperone function of Hfq in bacterial genera and illuminates how Sm proteins may evolve new functions.

The SmAP2 RNA binding motif in the 3'UTR affects mRNA stability in the crenarchaeum Sulfolobus solfataricus.

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism in Pro- and Eukaryotes. In this study, a collection of 53 mRNAs that co-purified with Sulfolobus solfataricus (Sso) SmAP2 were surveyed for a specific RNA binding motif (RBM). SmAP2 was shown to bind with high affinity to the deduced consensus RNA binding motif (SmAP2-cRBM) in vitro. Residues in SmAP2 interacting with the SmAP2-cRBM were mapped by UV-induced crosslinking in combination with mass-spectrometry, and verified by mutational analyses. The RNA-binding site on SmAP2 includes a modified uracil binding pocket containing a unique threonine (T40) located on the L3 face and a second residue, K25, located in the pore. To study the function of the SmAP2-RBM in vivo, three authentic RBMs were inserted in the 3′UTR of a lacS reporter gene. The presence of the SmAP2-RBM in the reporter-constructs resulted in decreased LacS activity and reduced steady state levels of lacS mRNA. Moreover, the presence of the SmAP2-cRBM in and the replacement of the lacS 3′UTR with that of Sso2194 encompassing a SmAP2-RBM apparently impacted on the stability of the chimeric transcripts. These results are discussed in light of the function(s) of eukaryotic Lsm proteins in RNA turnover.

Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation.

Specialized assembly factors facilitate the formation of many macromolecular complexes in vivo. The formation of Sm core structures of spliceosomal U-rich small nuclear ribonucleoprotein particles (UsnRNPs) requires assembly factors united in protein arginine methyltransferase 5 (PRMT5) and survival motor neuron (SMN) complexes. We demonstrate that perturbations of this assembly machinery trigger complex cellular responses that prevent aggregation of unassembled Sm proteins. Inactivation of the SMN complex results in the initial tailback of Sm proteins on the PRMT5 complex, followed by down-regulation of their encoding mRNAs. In contrast, reduction of pICln, a PRMT5 complex subunit, leads to the retention of newly synthesized Sm proteins on ribosomes and their subsequent lysosomal degradation. Overexpression of Sm proteins under these conditions results in a surplus of Sm proteins over pICln, promoting their aggregation. Our studies identify an elaborate safeguarding system that prevents individual Sm proteins from aggregating, contributing to cellular UsnRNP homeostasis.

Spinal Muscular Atrophy: From Defective Chaperoning of snRNP Assembly to Neuromuscular Dysfunction.

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder that results from decreased levels of the survival motor neuron (SMN) protein. SMN is part of a multiprotein complex that also includes Gemins 2–8 and Unrip. The SMN-Gemins complex cooperates with the protein arginine methyltransferase 5 (PRMT5) complex, whose constituents include WD45, PRMT5 and pICln. Both complexes function as molecular chaperones, interacting with and assisting in the assembly of an Sm protein core onto small nuclear RNAs (snRNAs) to generate small nuclear ribonucleoproteins (snRNPs), which are the operating components of the spliceosome. Molecular and structural studies have refined our knowledge of the key events taking place within the crowded environment of cells and the numerous precautions undertaken to ensure the faithful assembly of snRNPs. Nonetheless, it remains unclear whether a loss of chaperoning in snRNP assembly, considered as a “housekeeping” activity, is responsible for the selective neuromuscular phenotype in SMA. This review thus shines light on in vivo studies that point toward disturbances in snRNP assembly and the consequential transcriptome abnormalities as the primary drivers of the progressive neuromuscular degeneration underpinning the disease. Disruption of U1 snRNP or snRNP assembly factors other than SMN induces phenotypes that mirror aspects of SMN deficiency, and splicing defects, described in numerous SMA models, can lead to a DNA damage and stress response that compromises the survival of the motor system. Restoring the correct chaperoning of snRNP assembly is therefore predicted to enhance the benefit of SMA therapeutic modalities based on augmenting SMN expression.

An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing.

Archaeal Lsm rings as stable self-assembling tectons for protein nanofabrication

We have exploited the self-assembling properties of archaeal-derived protein Lsmα to generate new supramolecular forms based on its stable ring-shaped heptamer. We show that engineered ring tectons incorporating cysteine sidechains on obverse faces of the Lsmα7 toroid are capable of forming paired and stacked formations. A Cys-modified construct, N10C/E61C-Lsmα, appears to organize into disulfide-mediated tube formations up to 45 nm in length. We additionally report fabrication of cage-like protein clusters through conjugation of Cu2+ to His-tagged variants of the Lsmα7 tecton. These 400 kDa protein capsules are seen as cube particles with visible pores, and are reversibly dissembled into their component ring tectons by EDTA. The β-rich Lsmα supramolecular assemblies described are amenable to further fusion modifications, or for surface attachment, so providing potential for future applications that exploit the RNA-binding capacity of Lsm proteins, such as sensing applications.

The SmAP1/2 proteins of the crenarchaeon Sulfolobus solfataricus interact with the exosome and stimulate A-rich tailing of transcripts.

The conserved Sm and Sm-like proteins are involved in different aspects of RNA metabolism. Here, we explored the interactome of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso) to shed light on their physiological function(s). Both, SmAP1 and SmAP2 co-purified with several proteins involved in RNA-processing/modification, translation and protein turnover as well as with components of the exosome involved in 3΄ to 5΄ degradation of RNA. In follow-up studies a direct interaction with the poly(A) binding and accessory exosomal subunit DnaG was demonstrated. Moreover, elevated levels of both SmAPs resulted in increased abundance of the soluble exosome fraction, suggesting that they affect the subcellular localization of the exosome in the cell. The increased solubility of the exosome was accompanied by augmented levels of RNAs with A-rich tails that were further characterized using RNASeq. Hence, the observation that the Sso SmAPs impact on the activity of the exosome revealed a hitherto unrecognized function of SmAPs in archaea.

An activated Q-SNARE/SM protein complex as a possible intermediate in SNARE assembly.

Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18-1 as a possible acceptor complex for the R-SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18-1 remains tethered by the N-terminal domain of syntaxin1. Intriguingly, only one of the synaptobrevin truncation mutants (Syb1-65) was able to bind to the syntaxin1:SNAP25:Munc18-1 complex, suggesting either a cooperative zippering mechanism that proceeds bidirectionally or the progressive R-SNARE binding via an SM template. Moreover, the complex is resistant to disassembly by NSF Based on these findings, weconsider the ternary complex as a strong candidate for a physiological intermediate in SNARE assembly.