Structural insights into the cross-exon to cross-intron spliceosome switch.

Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron1. Alternatively, it can occur through an exon-defined pathway2-5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5' splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10-16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5' splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3' splice site usage. Together, our results highlight the role of the 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.

Structural basis of U12-type intron engagement by the fully assembled human minor spliceosome.

The minor spliceosome, which is responsible for the splicing of U12-type introns, comprises five small nuclear RNAs (snRNAs), of which only one is shared with the major spliceosome. In this work, we report the 3.3-angstrom cryo-electron microscopy structure of the fully assembled human minor spliceosome pre-B complex. The atomic model includes U11 small nuclear ribonucleoprotein (snRNP), U12 snRNP, and U4atac/U6atac.U5 tri-snRNP. U11 snRNA is recognized by five U11-specific proteins (20K, 25K, 35K, 48K, and 59K) and the heptameric Sm ring. The 3' half of the 5'-splice site forms a duplex with U11 snRNA; the 5' half is recognized by U11-35K, U11-48K, and U11 snRNA. Two proteins, CENATAC and DIM2/TXNL4B, specifically associate with the minor tri-snRNP. A structural analysis uncovered how two conformationally similar tri-snRNPs are differentiated by the minor and major prespliceosomes for assembly.

SMN regulates GEMIN5 expression and acts as a modifier of GEMIN5-mediated neurodegeneration.

GEMIN5 is essential for core assembly of small nuclear Ribonucleoproteins (snRNPs), the building blocks of spliceosome formation. Loss-of-function mutations in GEMIN5 lead to a neurodevelopmental syndrome among patients presenting with developmental delay, motor dysfunction, and cerebellar atrophy by perturbing SMN complex protein expression and assembly. Currently, molecular determinants of GEMIN5-mediated disease have yet to be explored. Here, we identified SMN as a genetic suppressor of GEMIN5-mediated neurodegeneration in vivo. We discovered that an increase in SMN expression by either SMN gene therapy replacement or the antisense oligonucleotide (ASO), Nusinersen, significantly upregulated the endogenous levels of GEMIN5 in mammalian cells and mutant GEMIN5-derived iPSC neurons. Further, we identified a strong functional association between the expression patterns of SMN and GEMIN5 in patient Spinal Muscular Atrophy (SMA)-derived motor neurons harboring loss-of-function mutations in the SMN gene. Interestingly, SMN binds to the C-terminus of GEMIN5 and requires the Tudor domain for GEMIN5 binding and expression regulation. Finally, we show that SMN upregulation ameliorates defective snRNP biogenesis and alternative splicing defects caused by loss of GEMIN5 in iPSC neurons and in vivo. Collectively, these studies indicate that SMN acts as a regulator of GEMIN5 expression and neuropathologies.

Global profiling identifies a stress-responsive tyrosine site on EDC3 regulating biomolecular condensate formation.

RNA granules are cytoplasmic condensates that organize biochemical and signaling complexes in response to cellular stress. Functional proteomic investigations under RNA-granule-inducing conditions are needed to identify protein sites involved in coupling stress response with ribonucleoprotein regulation. Here, we apply chemical proteomics using sulfonyl-triazole (SuTEx) probes to capture cellular responses to oxidative and nutrient stress. The stress-responsive tyrosine and lysine sites detected mapped to known proteins involved in processing body (PB) and stress granule (SG) pathways, including LSM14A, FUS, and Enhancer of mRNA-decapping protein 3 (EDC3). Notably, disruption of EDC3 tyrosine 475 (Y475) resulted in hypo-phosphorylation at S161 and S131 and altered protein-protein interactions (PPIs) with decapping complex components (DDX6, DCP1A/B) and 14-3-3 proteins. This resulting mutant form of EDC3 was capable of rescuing the PB-deficient phenotype of EDC3 knockout cells. Taken together, our findings identify Y475 as an arsenic-responsive site that regulates RNA granule formation by coupling EDC3 post-translational modification and PPI states.

Disrupting the Cdk9/Cyclin T1 heterodimer of 7SK snRNP for the Brd4 and AFF1/4 guided reconstitution of active P-TEFb.

P-TEFb modulates RNA polymerase II elongation through alternative interaction with negative and positive regulation factors. While inactive P-TEFbs are mainly sequestered in the 7SK snRNP complex in a chromatin-free state, most of its active forms are in complex with its recruitment factors, Brd4 and SEC, in a chromatin-associated state. Thus, switching from inactive 7SK snRNP to active P-TEFb (Brd4/P-TEFb or SEC/P-TEFb) is essential for global gene expression. Although it has been shown that cellular signaling stimulates the disruption of 7SK snRNP, releasing dephosphorylated and catalytically inactive P-TEFb, little is known about how the inactive released P-TEFb is reactivated. Here, we show that the Cdk9/CycT1 heterodimer released from 7SK snRNP is completely dissociated into monomers in response to stress. Brd4 or SEC then recruits monomerized Cdk9 and CycT1 to reassemble the core P-TEFb. Meanwhile, the binding of monomeric dephosphorylated Cdk9 to either Brd4 or SEC induces the autophosphorylation of T186 of Cdk9. Finally, the same mechanism is employed during nocodazole released entry into early G1 phase of cell cycle. Therefore, our studies demonstrate a novel mechanism by which Cdk9 and CycT1 monomers are reassembled on chromatin to form active P-TEFb by its interaction with Brd4 or SEC to regulate transcription.

CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.

TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation.

U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before the next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.

Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2.

Brr2 is an essential Ski2-like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N-terminal helicase cassette and a structurally similar but enzymatically inactive C-terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide-binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide-binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C-terminal cassette is more than two orders of magnitude higher than that of the active N-terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C-terminal cassette reduces nucleotide binding at the N-terminal cassette 70 Å away. Molecular dynamics simulations identified structural communication lines that likely mediate these long-range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicing.

psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation.

RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3'-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.

High-resolution structure of eukaryotic Fibrillarin interacting with Nop56 amino-terminal domain.

Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes.

Quality-Control Mechanism for Telomerase RNA Folding in the Cell.

Long non-coding RNAs can often fold into different conformations. Telomerase RNA, an essential component of the telomerase ribonucleoprotein (RNP) enzyme, must fold into a defined structure to fulfill its function with the protein catalytic subunit (TERT) and other accessory factors. However, the mechanism by which the correct folding of telomerase RNA is warranted in a cell is still unknown. Here we show that La-related protein Pof8 specifically recognizes the conserved pseudoknot region of telomerase RNA and instructs the binding of the Lsm2-8 complex to its mature 3' end, thus selectively protecting the correctly folded RNA from exonucleolytic degradation. In the absence of Pof8, TERT assembles with misfolded RNA and produces little telomerase activity. Therefore, Pof8 plays a key role in telomerase RNA folding quality control, ensuring that TERT only assembles with functional telomerase RNA to form active telomerase. Our finding reveals a mechanism for non-coding RNA folding quality control.

Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency.

A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.

Emerging Roles of Gemin5: From snRNPs Assembly to Translation Control.

RNA-binding proteins (RBPs) play a pivotal role in the lifespan of RNAs. The disfunction of RBPs is frequently the cause of cell disorders which are incompatible with life. Furthermore, the ordered assembly of RBPs and RNAs in ribonucleoprotein (RNP) particles determines the function of biological complexes, as illustrated by the survival of the motor neuron (SMN) complex. Defects in the SMN complex assembly causes spinal muscular atrophy (SMA), an infant invalidating disease. This multi-subunit chaperone controls the assembly of small nuclear ribonucleoproteins (snRNPs), which are the critical components of the splicing machinery. However, the functional and structural characterization of individual members of the SMN complex, such as SMN, Gemin3, and Gemin5, have accumulated evidence for the additional roles of these proteins, unveiling their participation in other RNA-mediated events. In particular, Gemin5 is a multidomain protein that comprises tryptophan-aspartic acid (WD) repeat motifs at the N-terminal region, a dimerization domain at the middle region, and a non-canonical RNA-binding domain at the C-terminal end of the protein. Beyond small nuclear RNA (snRNA) recognition, Gemin5 interacts with a selective group of mRNA targets in the cell environment and plays a key role in reprogramming translation depending on the RNA partner and the cellular conditions. Here, we review recent studies on the SMN complex, with emphasis on the individual components regarding their involvement in cellular processes critical for cell survival.

snoRNPs: Functions in Ribosome Biogenesis.

Ribosomes are perhaps the most critical macromolecular machine as they are tasked with carrying out protein synthesis in cells. They are incredibly complex structures composed of protein components and heavily chemically modified RNAs. The task of assembling mature ribosomes from their component parts consumes a massive amount of energy and requires greater than 200 assembly factors. Among the most critical of these are small nucleolar ribonucleoproteins (snoRNPs). These are small RNAs complexed with diverse sets of proteins. As suggested by their name, they localize to the nucleolus, the site of ribosome biogenesis. There, they facilitate multiple roles in ribosomes biogenesis, such as pseudouridylation and 2'-O-methylation of ribosomal (r)RNA, guiding pre-rRNA processing, and acting as molecular chaperones. Here, we reviewed their activity in promoting the assembly of ribosomes in eukaryotes with regards to chemical modification and pre-rRNA processing.

Zmat2 in mammals: conservation and diversification among genes and Pseudogenes.

BACKGROUND: Recent advances in genetics and genomics present unique opportunities for enhancing our understanding of mammalian biology and evolution through detailed multi-species comparative analysis of gene organization and expression. Yet, of the more than 20,000 protein coding genes found in mammalian genomes, fewer than 10% have been examined in any detail. Here we elucidate the power of data available in publicly-accessible genomic and genetic resources by querying them to evaluate Zmat2, a minimally studied gene whose human ortholog has been implicated in spliceosome function and in keratinocyte differentiation. RESULTS: We find extensive conservation in coding regions and overall structure of Zmat2 in 18 mammals representing 13 orders and spanning ~ 165 million years of evolutionary development, and in their encoded proteins. We identify a tandem duplication in the Zmat2 gene and locus in opossum, but not in other monotremes, marsupials, or other mammals, indicating that this event occurred subsequent to the divergence of these species from one another. We also define a collection of Zmat2 pseudogenes in half of the mammals studied, and suggest based on phylogenetic analysis that they each arose independently in the recent evolutionary past. CONCLUSIONS: Mammalian Zmat2 genes and ZMAT2 proteins illustrate conservation of structure and sequence, along with the development and diversification of pseudogenes in a large fraction of species. Collectively, these observations also illustrate how the focused identification and interpretation of data found in public genomic and gene expression resources can be leveraged to reveal new insights of potentially high biological significance.

NHP2 deficiency impairs rRNA biogenesis and causes pulmonary fibrosis and Høyeraal-Hreidarsson syndrome.

Telomeres are nucleoprotein structures at the end of chromosomes. The telomerase complex, constituted of the catalytic subunit TERT, the RNA matrix hTR and several cofactors, including the H/ACA box ribonucleoproteins Dyskerin, NOP10, GAR1, NAF1 and NHP2, regulates telomere length. In humans, inherited defects in telomere length maintenance are responsible for a wide spectrum of clinical premature aging manifestations including pulmonary fibrosis (PF), dyskeratosis congenita (DC), bone marrow failure and predisposition to cancer. NHP2 mutations have been so far reported only in two patients with DC. Here, we report the first case of Høyeraal-Hreidarsson syndrome, the severe form of DC, caused by biallelic missense mutations in NHP2. Additionally, we identified three unrelated patients with PF carrying NHP2 heterozygous mutations. Strikingly, one of these patients acquired a somatic mutation in the promoter of TERT that likely conferred a selective advantage in a subset of blood cells. Finally, we demonstrate that a functional deficit of human NHP2 affects ribosomal RNA biogenesis. Together, our results broaden the functional consequences and clinical spectrum of NHP2 deficiency.

The inactive C-terminal cassette of the dual-cassette RNA helicase BRR2 both stimulates and inhibits the activity of the N-terminal helicase unit.

The RNA helicase bad response to refrigeration 2 homolog (BRR2) is required for the activation of the spliceosome before the first catalytic step of RNA splicing. BRR2 represents a distinct subgroup of Ski2-like nucleic acid helicases whose members comprise tandem helicase cassettes. Only the N-terminal cassette of BRR2 is an active ATPase and can unwind substrate RNAs. The C-terminal cassette represents a pseudoenzyme that can stimulate RNA-related activities of the N-terminal cassette. However, the molecular mechanisms by which the C-terminal cassette modulates the activities of the N-terminal unit remain elusive. Here, we show that N- and C-terminal cassettes adopt vastly different relative orientations in a crystal structure of BRR2 in complex with an activating domain of the spliceosomal Prp8 protein at 2.4 Å resolution compared with the crystal structure of BRR2 alone. Likewise, inspection of BRR2 structures within spliceosomal complexes revealed that the cassettes occupy different relative positions and engage in different intercassette contacts during different splicing stages. Engineered disulfide bridges that locked the cassettes in two different relative orientations had opposite effects on the RNA-unwinding activity of the N-terminal cassette, with one configuration enhancing and the other configuration inhibiting RNA unwinding compared with the unconstrained protein. Moreover, we found that differences in relative positioning of the cassettes strongly influence RNA-stimulated ATP hydrolysis by the N-terminal cassette. Our results indicate that the inactive C-terminal cassette of BRR2 can both positively and negatively affect the activity of the N-terminal helicase unit from a distance.

Structural basis for the dimerization of Gemin5 and its role in protein recruitment and translation control.

In all organisms, a selected type of proteins accomplishes critical roles in cellular processes that govern gene expression. The multifunctional protein Gemin5 cooperates in translation control and ribosome binding, besides acting as the RNA-binding protein of the survival of motor neuron (SMN) complex. While these functions reside on distinct domains located at each end of the protein, the structure and function of the middle region remained unknown. Here, we solved the crystal structure of an extended tetratricopeptide (TPR)-like domain in human Gemin5 that self-assembles into a previously unknown canoe-shaped dimer. We further show that the dimerization module is functional in living cells driving the interaction between the viral-induced cleavage fragment p85 and the full-length Gemin5, which anchors splicing and translation members. Disruption of the dimerization surface by a point mutation in the TPR-like domain prevents this interaction and also abrogates translation enhancement induced by p85. The characterization of this unanticipated dimerization domain provides the structural basis for a role of the middle region of Gemin5 as a central hub for protein-protein interactions.

Negative cooperativity between Gemin2 and RNA provides insights into RNA selection and the SMN complex's release in snRNP assembly.

The assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around the nonameric Sm site of snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. SMN deficiency causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores is largely unknown, especially how the SMN complex achieves high RNA assembly specificity and how it is released. Here we show, using crystallographic and biochemical approaches, that Gemin2 of the SMN complex enhances RNA specificity of SmD1/D2/F/E/G via a negative cooperativity between Gemin2 and RNA in binding SmD1/D2/F/E/G. Gemin2, independent of its N-tail, constrains the horseshoe-shaped SmD1/D2/F/E/G from outside in a physiologically relevant, narrow state, enabling high RNA specificity. Moreover, the assembly of RNAs inside widens SmD1/D2/F/E/G, causes the release of Gemin2/SMN allosterically and allows SmD3/B to join. The assembly of SmD3/B further facilitates the release of Gemin2/SMN. This is the first to show negative cooperativity in snRNP assembly, which provides insights into RNA selection and the SMN complex's release. These findings reveal a basic mechanism of snRNP core assembly and facilitate pathogenesis studies of SMA.