Sm Proteins Research Articles

Membrane fusion reactions limited by defective SNARE zippering or stiff lipid fatty acyl composition have distinct requirements for Sec17, Sec18, and adenine nucleotide.

Intracellular membrane fusion is catalyzed by SNAREs, Rab GTPases, SM proteins, tethers, Sec18/NSF and Sec17/SNAP. Membrane fusion has been reconstituted with purified vacuolar proteins and lipids to address 3 salient questions: whether ATP hydrolysis by Sec18 affects its promotion of fusion, whether fusion promotion by Sec17 and Sec18 is only seen with mutant SNAREs or can also be seen with wild-type SNAREs, and whether Sec17 and Sec18 only promote fusion when they work together or whether they can each work separately. Fusion is driven by two engines, completion of SNARE zippering (which does not need Sec17/Sec18) and Sec17/Sec18-mediated fusion (needing SNAREs but not the energy from their complete zippering). Sec17 is required to rescue fusion that is blocked by incomplete zippering, though optimal rescue also needs the ATPase Sec18. ATP is an essential Sec18 ligand, but at limiting Sec17 levels Sec18 ATP hydrolysis also drives release of Sec17 without concomitant trans-SNARE complex disassembly. At higher (physiological) Sec17 levels, or without ATP hydrolysis, fusion prevails over Sec17 release. Stiff 16:0, 18:1 fatty acyl chain lipids provide an alternative route to suppressing fusion, with entirely wild-type SNAREs and without impediment to zippering. In this case, Sec17 and Sec18 restore comparable fusion with either ATP or a nonhydrolyzable analog. Fusion blocked by impaired zippering can be restored by concentrated Sec17 alone (but not by Sec18), while fusion inhibited by stiff fatty acyl chains is partially restored by Sec18 alone (but not by Sec17). With distinct fusion impediments, Sec18 and Sec17 have both shared roles and independent roles in promoting fusion.

Productive mRNA Chromatin Escape is Promoted by PRMT5 Methylation of SNRPB.

Protein Arginine Methyltransferase 5 (PRMT5) regulates RNA splicing and transcription by symmetric dimethylation of arginine residues (Rme2s/SDMA) in many RNA binding proteins. However, the mechanism by which PRMT5 couples splicing to transcriptional output is unknown. Here, we demonstrate that a major function of PRMT5 activity is to promote chromatin escape of a novel, large class of mRNAs that we term Genomically Retained Incompletely Processed Polyadenylated Transcripts (GRIPPs). Using nascent and total transcriptomics, spike-in controlled fractionated cell transcriptomics, and total and fractionated cell proteomics, we show that PRMT5 inhibition and knockdown of the PRMT5 SNRP (Sm protein) adapter protein pICln (CLNS1A) -but not type I PRMT inhibition-leads to gross detention of mRNA, SNRPB, and SNRPD3 proteins on chromatin. Compared to most transcripts, these chromatin-trapped polyadenylated RNA transcripts have more introns, are spliced slower, and are enriched in detained introns. Using a combination of PRMT5 inhibition and inducible isogenic wildtype and arginine-mutant SNRPB, we show that arginine methylation of these snRNPs is critical for mediating their homeostatic chromatin and RNA interactions. Overall, we conclude that a major role for PRMT5 is in controlling transcript processing and splicing completion to promote chromatin escape and subsequent nuclear export.

Intron Retention of DDX39A Driven by SNRPD2 is a Crucial Splicing Axis for Oncogenic MYC/Spliceosome Program in Hepatocellular Carcinoma.

RNA splicing is a dynamic molecular process in response to environmental stimuli and is strictly regulated by the spliceosome. Sm proteins, constituents of the spliceosome, are key components that mediate splicing reactions; however, their potential role in hepatocellular carcinoma (HCC) is poorly understood. In the study, SNRPD2 (PD2) is found to be the most highly upregulated Sm protein in HCC and to act as an oncogene. PD2 modulates DDX39A intron retention together with HNRNPL to sustain the DDX39A short variant (39A_S) expression. Mechanistically, 39A_S can mediate MYC mRNA nuclear export to maintain high MYC protein expression, while MYC in turn potentiates PD2 transcription. Importantly, digitoxin can directly interact with PD2 and has a notable cancer-suppressive effect on HCC. The study reveals a novel mechanism by which DDX39A senses oncogenic MYC signaling and undergoes splicing via PD2 to form a positive feedback loop in HCC, which can be targeted by digitoxin.

Effect of E134K pathogenic mutation of SMN protein on SMN-SmD1 interaction, with implication in spinal muscular atrophy: A molecular dynamics study

Spinal muscular atrophy (SMA) is a disease that results from mutations in the Survival of Motor Neuron (SMN) gene 1, leading to muscle atrophy due to motor neurons degeneration. SMN plays a crucial role in the assembly of spliceosomal small nuclear ribonucleoprotein complexes via binding to the arginine-glycine rich C-terminal tails of Sm proteins recognized by SMN Tudor domain. E134K Tudor mutation, cause of the more severe type I SMA, compromises the SMN-Sm interaction without a perturbation of the domain fold. By molecular dynamics simulations, we investigated the mechanism of Tudor-SmD1 interaction, and the effects on it of E134K mutation. It was observed that E134 is crucial to catch the positive dimethylated arginines (DMRs) of the SmD1 tail that, wrapping around the acidic Tudor surface, enters a central DMR into an aromatic cage. The flexible cage residue Y130 must be blocked from the wrapped tail to assure a stable binding.The charge inversion in E134K mutation causes the loss of a critical anchor point, disfavoring the tail wrapping and leaving Y130 free to swing, leading to DMR detachments and exposition of the C-terminal region of the tail. This could suggest new hypotheses regarding a possible autoimmune response by anti-Sm autoantibodies.

B-cell immune deficiency in twin sisters expands the phenotype of MOPDI.

Microcephalic osteodysplastic primordial dwarfism type I (MOPDI) is a very rare and severe autosomal recessive disorder characterized by marked intrauterine growth retardation, skeletal dysplasia, microcephaly and brain malformations. MOPDI is caused by biallelic mutations in RNU4ATAC, a non-coding gene involved in U12-type splicing of 1% of the introns in the genome, which are recognized by their specific splicing consensus sequences. Here, we describe a unique observation of immunodeficiency in twin sisters with mild MOPDI, who harbor a novel n.108_126del mutation, encompassing part of the U4atac snRNA 3' stem-loop and Sm protein binding site, and the previously reported n.111G>A mutation. Interestingly, both twin sisters show mild B-cell anomalies, including low naive B-cell counts and increased memory B-cell and plasmablasts counts, suggesting partial and transitory blockage of B-cell maturation and/or excessive activation of naive B-cells. Hence, the localization of a mutation in stem II of U4atac snRNA, as observed in another RNU4ATAC-opathy with immunodeficiency, that is, Roifman syndrome (RFMN), is not required for the occurrence of an immune deficiency. Finally, we emphasize the importance of considering immunodeficiency in MOPDI management to reduce the risk of serious infectious episodes.

Sec18 binds the tethering/SM complex HOPS to engage the Qc-SNARE for membrane fusion.

Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in trans, impaired Qc recognition by limiting HOPS without Sec18 still allows substantial trans-SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.

SM protein Sly1 and a SNARE Habc domain promote membrane fusion through multiple mechanisms.

SM proteins including Sly1 are essential cofactors of SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants and chemically defined in vitro assays, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and that close-range tethering promotes trans-complex assembly when cis-SNARE assembly is a competing process. Further, we demonstrate that the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: it is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. "Split Sed5," with Habc presented solely as a soluble fragment, can function both in vitro and in vivo. Habc appears to facilitate events leading to lipid mixing rather than promoting opening or stability of the fusion pore.

Cancer-associated SNRPD3 mutation confers resistance to hypoxia, which is attenuated by DRP1 inhibition

RNA splicing is a fundamental cellular mechanism performed by spliceosomes that synthesise multiple mature RNA isoforms from a single gene. The association between spliceosome abnormality and solid cancers remains largely unknown. Here, we demonstrated that Sm proteins, which are common components of the spliceosomes and constitute the Sm ring, were overexpressed in multiple cancers and their expression levels were correlated with clinical prognosis. In a pan-cancer mutational hotspot in the Sm ring at SNRPD3 G96V, we found that the G96V substitution confers resistance to hypoxia. RNA-seq detected numerous differentially spliced events between the wild-type and mutation-carrying cells cultured under hypoxia, wherein skipping exons and mutually exclusive exons were frequently observed. This was observed in DNM1L mRNA, which encodes the DRP1 protein that regulates mitochondrial fission. The mitochondria of cells carrying this mutation were excessively fragmented compared with those of wild-type cells. Furthermore, treatment with a DRP1 inhibitor (Mdivi-1) recovered the over-fragmented mitochondria, leading to the attenuation of hypoxia resistance in the mutant cells. These results propose a novel correlation between the cancer-related spliceosome abnormality and mitochondrial fission. Thus, targeting SNRPD3 G96V with a DRP1 inhibitor is a potential treatment strategy for cancers with spliceosome abnormalities.

Analysis of Lsm Protein-Mediated Regulation in the Haloarchaeon Haloferax mediterranei.

The Sm protein superfamily includes Sm, like-Sm (Lsm), and Hfq found in the Eukarya, Archaea, and Bacteria domains. Archaeal Lsm proteins have been shown to bind sRNAs and are probably involved in various cellular processes, suggesting a similar function in regulating sRNAs by Hfq in bacteria. Moreover, archaeal Lsm proteins probably represent the ancestral Lsm domain from which eukaryotic Sm proteins have evolved. In this work, Haloferax mediterranei was used as a model organism because it has been widely used to investigate the nitrogen cycle and its regulation in Haloarchaea. Predicting this protein's secondary and tertiary structures has resulted in a three-dimensional model like the solved Lsm protein structure of Archaeoglobus fulgidus. To obtain information on the oligomerization state of the protein, homologous overexpression and purification by means of molecular exclusion chromatography have been performed. The results show that this protein can form hexameric complexes, which can aggregate into 6 or 12 hexameric rings depending on the NaCl concentration and without RNA. In addition, the study of transcriptional expression via microarrays has allowed us to obtain the target genes regulated by the Lsm protein under nutritional stress conditions: nitrogen or carbon starvation. Microarray analysis has shown the first universal stress proteins (USP) in this microorganism that mediate survival in situations of nitrogen deficiency.

The Impact of p70S6 Kinase-Dependent Phosphorylation of Gemin2 in UsnRNP Biogenesis.

The survival motor neuron (SMN) complex is a multi-megadalton complex involved in post-transcriptional gene expression in eukaryotes via promotion of the biogenesis of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). The functional center of the complex is formed from the SMN/Gemin2 subunit. By binding the pentameric ring made up of the Sm proteins SmD1/D2/E/F/G and allowing for their transfer to a uridine-rich short nuclear RNA (UsnRNA), the Gemin2 protein in particular is crucial for the selectivity of the Sm core assembly. It is well established that post-translational modifications control UsnRNP biogenesis. In our work presented here, we emphasize the crucial role of Gemin2, showing that the phospho-status of Gemin2 influences the capacity of the SMN complex to condense in Cajal bodies (CBs) in vivo. Additionally, we define Gemin2 as a novel and particular binding partner and phosphorylation substrate of the mTOR pathway kinase ribosomal protein S6 kinase beta-1 (p70S6K). Experiments using size exclusion chromatography further demonstrated that the Gemin2 protein functions as a connecting element between the 6S complex and the SMN complex. As a result, p70S6K knockdown lowered the number of CBs, which in turn inhibited in vivo UsnRNP synthesis. In summary, these findings reveal a unique regulatory mechanism of UsnRNP biogenesis.

The SMN complex drives structural changes in human snRNAs to enable snRNP assembly

Spliceosomal snRNPs are multicomponent particles that undergo a complex maturation pathway. Human Sm-class snRNAs are generated as 3′-end extended precursors, which are exported to the cytoplasm and assembled together with Sm proteins into core RNPs by the SMN complex. Here, we provide evidence that these pre-snRNA substrates contain compact, evolutionarily conserved secondary structures that overlap with the Sm binding site. These structural motifs in pre-snRNAs are predicted to interfere with Sm core assembly. We model structural rearrangements that lead to an open pre-snRNA conformation compatible with Sm protein interaction. The predicted rearrangement pathway is conserved in Metazoa and requires an external factor that initiates snRNA remodeling. We show that the essential helicase Gemin3, which is a component of the SMN complex, is crucial for snRNA structural rearrangements during snRNP maturation. The SMN complex thus facilitates ATP-driven structural changes in snRNAs that expose the Sm site and enable Sm protein binding.

The archaeal Lsm protein from Pyrococcus furiosus binds co-transcriptionally to poly(U)-rich target RNAs.

Posttranscriptional processes in Bacteria include the association of small regulatory RNAs (sRNA) with a target mRNA. The sRNA/mRNA annealing process is often mediated by an RNA chaperone called Hfq. The functional role of bacterial and eukaryotic Lsm proteins is partially understood, whereas knowledge about archaeal Lsm proteins is scarce. Here, we used the genetically tractable archaeal hyperthermophile Pyrococcus furiosus to identify the protein interaction partners of the archaeal Sm-like proteins (PfuSmAP1) using mass spectrometry and performed a transcriptome-wide binding site analysis of PfuSmAP1. Most of the protein interaction partners we found are part of the RNA homoeostasis network in Archaea including ribosomal proteins, the exosome, RNA-modifying enzymes, but also RNA polymerase subunits, and transcription factors. We show that PfuSmAP1 preferentially binds messenger RNAs and antisense RNAs recognizing a gapped poly(U) sequence with high affinity. Furthermore, we found that SmAP1 co-transcriptionally associates with target RNAs. Our study reveals that in contrast to bacterial Hfq, PfuSmAP1 does not affect the transcriptional activity or the pausing behaviour of archaeal RNA polymerases. We propose that PfuSmAP1 recruits antisense RNAs to target mRNAs and thereby executes its putative regulatory function on the posttranscriptional level.

In vitro methylation of the U7 snRNP subunits Lsm11 and SmE by the PRMT5/MEP50/pICln methylosome.

U7 snRNP is a multisubunit endonuclease required for 3' end processing of metazoan replication-dependent histone pre-mRNAs. In contrast to the spliceosomal snRNPs, U7 snRNP lacks the Sm subunits D1 and D2 and instead contains two related proteins, Lsm10 and Lsm11. The remaining five subunits of the U7 heptameric Sm ring, SmE, F, G, B, and D3, are shared with the spliceosomal snRNPs. The pathway that assembles the unique ring of U7 snRNP is unknown. Here, we show that a heterodimer of Lsm10 and Lsm11 tightly interacts with the methylosome, a complex of the arginine methyltransferase PRMT5, MEP50, and pICln known to methylate arginines in the carboxy-terminal regions of the Sm proteins B, D1, and D3 during the spliceosomal Sm ring assembly. Both biochemical and cryo-EM structural studies demonstrate that the interaction is mediated by PRMT5, which binds and methylates two arginine residues in the amino-terminal region of Lsm11. Surprisingly, PRMT5 also methylates an amino-terminal arginine in SmE, a subunit that does not undergo this type of modification during the biogenesis of the spliceosomal snRNPs. An intriguing possibility is that the unique methylation pattern of Lsm11 and SmE plays a vital role in the assembly of the U7 snRNP.

NSF/αSNAP2-mediated cis-SNARE complex disassembly precedes vesicle fusion in Arabidopsis cytokinesis.

Eukaryotic membrane fusion requires trans-SNARE complexes bridging the gap between adjacent membranes1. Fusion between a transport vesicle and its target membrane transforms the trans- into a cis-SNARE complex. The latter interacts with the hexameric AAA+-ATPase N-ethylmaleimide-sensitive factor (NSF) and its co-factor alpha-soluble NSF attachment protein (αSNAP), forming a 20S complex2,3. ATPase activity disassembles the SNAP receptor (SNARE) complex into Qa-SNARE, which folds back onto itself, and its partners4,5. The fusion of identical membranes has a different sequence of events6. The fusion partners each have cis-SNARE complexes to be broken up by NSF and αSNAP. The Qa-SNARE monomers are then stabilized by interaction with Sec1/Munc18-type regulators (SM proteins) to form trans-SNARE complexes, as shown for the yeast vacuole7. Membrane fusion in Arabidopsis cytokinesis is formally akin to vacuolar fusion8. Membrane vesicles fuse with one another to form the partitioning membrane known as the cell plate. Cis-SNARE complexes of cytokinesis-specific Qa-SNARE KNOLLE and its SNARE partners are assembled at the endoplasmic reticulum and delivered by traffic via the Golgi/trans-Golgi network to the cell division plane9. The SM protein KEULE is required for the formation of trans-SNARE complexes between adjacent membrane vesicles10. Here we identify NSF and its adaptor αSNAP2 as necessary for the disassembly of KNOLLE cis-SNARE complexes, which is a prerequisite for KNOLLE-KEULE interaction in cytokinesis. In addition, we show that NSF is required for other trafficking pathways and interacts with the respective Q-SNAREs. The SNARE complex disassembly machinery is conserved in plants and plays a unique essential role in cytokinesis.

Comprehensive Bioinformatics Analysis of the Biodiversity of Lsm Proteins in the Archaea Domain.

The Sm protein superfamily includes Sm, like-Sm (Lsm), and Hfq proteins. Sm and Lsm proteins are found in the Eukarya and Archaea domains, respectively, while Hfq proteins exist in the Bacteria domain. Even though Sm and Hfq proteins have been extensively studied, archaeal Lsm proteins still require further exploration. In this work, different bioinformatics tools are used to understand the diversity and distribution of 168 Lsm proteins in 109 archaeal species to increase the global understanding of these proteins. All 109 archaeal species analyzed encode one to three Lsm proteins in their genome. Lsm proteins can be classified into two groups based on molecular weight. Regarding the gene environment of lsm genes, many of these genes are located adjacent to transcriptional regulators of the Lrp/AsnC and MarR families, RNA-binding proteins, and ribosomal protein L37e. Notably, only proteins from species of the class Halobacteria conserved the internal and external residues of the RNA-binding site identified in Pyrococcus abyssi, despite belonging to different taxonomic orders. In most species, the Lsm genes show associations with 11 genes: rpl7ae, rpl37e, fusA, flpA, purF, rrp4, rrp41, hel308, rpoD, rpoH, and rpoN. We propose that most archaeal Lsm proteins are related to the RNA metabolism, and the larger Lsm proteins could perform different functions and/or act through other mechanisms of action.

The cancer testis antigen TDRD1 regulates prostate cancer proliferation by associating with the snRNP biogenesis machinery.

Prostate cancer is the most commonly diagnosed noncutaneous cancer in American men. TDRD1, a germ cell-specific gene, is erroneously expressed in more than half of prostate tumors, but its role in prostate cancer development remains elusive. In this study, we identified a PRMT5-TDRD1 signaling axis that regulates the proliferation of prostate cancer cells. PRMT5 is a protein arginine methyltransferase essential for small nuclear ribonucleoprotein (snRNP) biogenesis. Methylation of Sm proteins by PRMT5 is a critical initiation step for assembling snRNPs in the cytoplasm, and the final snRNP assembly takes place in Cajal bodies in the nucleus. By mass spectrum analysis, we found that TDRD1 interacts with multiple subunits of the snRNP biogenesis machinery. In the cytoplasm, TDRD1 interacts with methylated Sm proteins in a PRMT5-dependent manner. In the nucleus, TDRD1 interacts with Coilin, the scaffold protein of Cajal bodies. Ablation of TDRD1 in prostate cancer cells disrupted the integrity of Cajal bodies, affected the snRNP biogenesis, and reduced cell proliferation. Taken together, this study represents the first characterization of TDRD1 functions in prostate cancer development and suggests TDRD1 as a potential therapeutic target for prostate cancer treatment.

Investigation of the potential off-target effects on Munc 18 (p67) involved in synaptic vesical transport due to the interaction with peptide p5.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and SM (Sec1/Munc-18 like) proteins are involved in the docking of vesicles with the target membranes followed by the release of neurotransmitters in the brain. The Syntaxin protein plays a crucial role in the SNARE complex formation. Munc 18 (p67) is known to regulate the different functions of these SNARE complexes by binding with different conformations of Syntaxin. It has also been established that the absence of p67 results in neuronal death. Our previous work studied the effect of the peptide p5, a promising drug candidate, on CDK5-p25, an established Alzheimer's disease pathological complex. In vivo and in vitro data shows that p5 selectively inhibits the pathological complex CDK5-p25 without affecting CDK5 physiology in presence of p67. This study focuses on p67-p5 interaction and on the characterization of any conformational changes induced by it, especially in the Syntaxin binding site (the cleft region between domains 1 and 3a). This provides insights into the potential off-target effects caused by p5 due to a potential inhibition of SNARE complex formation. Our Monte Carlo/Molecular dynamics method (MCMD) predicted nine different binding modes, four of which were analyzed previously and indicated conformational changes induced in the Syntaxin binding site at the important functional domains 3a and 2. Herein we investigate the remaining five p67-p5 modes in order to fully understand the interaction dynamics, and the relevant results are presented.

SNARE Proteins in Synaptic Vesicle Fusion.

Neurotransmitters are stored in small membrane-bound vesicles at synapses; a subset of synaptic vesicles is docked at release sites. Fusion of docked vesicles with the plasma membrane releases neurotransmitters. Membrane fusion at synapses, as well as all trafficking steps of the secretory pathway, is mediated by SNARE proteins. The SNAREs are the minimal fusion machinery. They zipper from N-termini to membrane-anchored C-termini to form a 4-helix bundle that forces the apposed membranes to fuse. At synapses, the SNAREs comprise a single helix from syntaxin and synaptobrevin; SNAP-25 contributes the other two helices to complete the bundle. Unc13 mediates synaptic vesicle docking and converts syntaxin into the permissive "open" configuration. The SM protein, Unc18, is required to initiate and proofread SNARE assembly. The SNAREs are then held in a half-zippered state by synaptotagmin and complexin. Calcium removes the synaptotagmin and complexin block, and the SNAREs drive vesicle fusion. After fusion, NSF and alpha-SNAP unwind the SNAREs and thereby recharge the system for further rounds of fusion. In this chapter, we will describe the discovery of the SNAREs, their relevant structural features, models for their function, and the central role of Unc18. In addition, we will touch upon the regulation of SNARE complex formation by Unc13, complexin, and synaptotagmin.

Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell

The spliceosome, responsible for all mature protein-coding transcripts of eukaryotic intron-containing genes, consists of small uridine-rich nuclear ribonucleoproteins (UsnRNPs). The assembly of UsnRNPs depends, on one hand, on the arginine methylation of Sm proteins catalyzed by the PRMT5 complex. On the other hand, it depends on the phosphorylation of the PRMT5 subunit pICln by the Uncoordinated Like Kinase 1 (ULK1). In consequence, phosphorylation of pICln affects the stability of the UsnRNP assembly intermediate, the so-called 6 S complex. The detailed mechanisms of phosphorylation-dependent integrity and subsequent UsnRNP assembly of the 6 S complex in vivo have not yet been analyzed.By using a phospho-specific antibody against ULK1-dependent phosphorylation sites of pICln, we visualize the intracellular distribution of phosphorylated pICln. Furthermore, we detect the colocaliphosphor-pICln1 with phospho-pICln by size-exclusion chromatography and immunofluorescence techniques. We also show that phosphorylated pICln is predominantly present in the 6 S complex. The addition of ULK1 to in vitro produced 6 S complex, as well as the reconstitution of ULK1 in ULK1-deficient cells, increases the efficiency of snRNP biogenesis. Accordingly, inhibition of ULK1 and the associated decreased pICln phosphorylation lead to accumulation of the 6 S complex and reduction in the spliceosomal activity of the cell.

PICLN modulates alternative splicing and light/temperature responses in plants.

Plants undergo transcriptome reprograming to adapt to daily and seasonal fluctuations in light and temperature conditions. While most efforts have focused on the role of master transcription factors, the importance of splicing factors modulating these processes is now emerging. Efficient pre-mRNA splicing depends on proper spliceosome assembly, which in plants and animals requires the methylosome complex. Ion Chloride nucleotide-sensitive protein (PICLN) is part of the methylosome complex in both humans and Arabidopsis (Arabidopsis thaliana), and we show here that the human PICLN ortholog rescues phenotypes of Arabidopsis picln mutants. Altered photomorphogenic and photoperiodic responses in Arabidopsis picln mutants are associated with changes in pre-mRNA splicing that partially overlap with those in PROTEIN ARGININE METHYL TRANSFERASE5 (prmt5) mutants. Mammalian PICLN also acts in concert with the Survival Motor Neuron (SMN) complex component GEMIN2 to modulate the late steps of UsnRNP assembly, and many alternative splicing events regulated by PICLN but not PRMT5, the main protein of the methylosome, are controlled by Arabidopsis GEMIN2. As with GEMIN2 and SM PROTEIN E1/PORCUPINE (SME1/PCP), low temperature, which increases PICLN expression, aggravates morphological and molecular defects of picln mutants. Taken together, these results establish a key role for PICLN in the regulation of pre-mRNA splicing and in mediating plant adaptation to daily and seasonal fluctuations in environmental conditions.