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.

Viruses and Cajal Bodies: A Critical Cellular Target in Virus Infection?

Nuclear bodies (NBs) are dynamic structures present in eukaryotic cell nuclei. They are not bounded by membranes and are often considered biomolecular condensates, defined structurally and functionally by the localisation of core components. Nuclear architecture can be reorganised during normal cellular processes such as the cell cycle as well as in response to cellular stress. Many plant and animal viruses target their proteins to NBs, in some cases triggering their structural disruption and redistribution. Although not all such interactions have been well characterised, subversion of NBs and their functions may form a key part of the life cycle of eukaryotic viruses that require the nucleus for their replication. This review will focus on Cajal bodies (CBs) and the viruses that target them. Since CBs are dynamic structures, other NBs (principally nucleoli and promyelocytic leukaemia, PML and bodies), whose components interact with CBs, will also be considered. As well as providing important insights into key virus-host cell interactions, studies on Cajal and associated NBs may identify novel cellular targets for development of antiviral compounds.

Nucleolar reorganization after cellular stress is orchestrated by SMN shuttling between nuclear compartments.

Spinal muscular atrophy is an autosomal recessive neuromuscular disease caused by mutations in the multifunctional protein Survival of Motor Neuron, or SMN. Within the nucleus, SMN localizes to Cajal bodies, which are associated with nucleoli, nuclear organelles dedicated to the first steps of ribosome biogenesis. The highly organized structure of the nucleolus can be dynamically altered by genotoxic agents. RNAP1, Fibrillarin, and nucleolar DNA are exported to the periphery of the nucleolus after genotoxic stress and, once DNA repair is fully completed, the organization of the nucleolus is restored. We find that SMN is required for the restoration of the nucleolar structure after genotoxic stress. During DNA repair, SMN shuttles from the Cajal bodies to the nucleolus. This shuttling is important for nucleolar homeostasis and relies on the presence of Coilin and the activity of PRMT1.

The Cajal body protein p80-coilin forms a complex with the adenovirus L4-22K protein and facilitates the nuclear export of adenovirus mRNA.

IMPORTANCE: The architecture of sub-nuclear structures of eucaryotic cells is often changed during the infectious cycle of many animal and plant viruses. Cajal bodies (CBs) form a major sub-nuclear structure whose functions may include the regulation of cellular RNA metabolism. During the lifecycle of human adenovirus 5 (Ad5), CBs are reorganized from their spherical-like structure into smaller clusters termed microfoci. The mechanism of this reorganization and its significance for virus replication has yet to be established. Here we show that the major CB protein, p80-coilin, facilitates the nuclear export of Ad5 transcripts. Depletion of p80-coilin by RNA interference led to lowered levels of viral proteins and infectious virus. p80-coilin was found to form a complex with the viral L4-22K protein in Ad5-infected cells and in some reorganized microfoci. These findings assign a new role for p80-coilin as a potential regulator of infection by a human DNA virus.

VRK1 variants at the cross road of Cajal body neuropathogenic mechanisms in distal neuropathies and motor neuron diseases.

Distal hereditary neuropathies and neuro motor diseases are complex neurological phenotypes associated with pathogenic variants in a large number of genes, but in some the origin is unknown. Recently, rare pathogenic variants of the human VRK1 gene have been associated with these neurological phenotypes. All VRK1 pathogenic variants are recessive, and their clinical presentation occurs in either homozygous or compound heterozygous patients. The pathogenic VRK1 gene pathogenic variants are located in three clusters within the protein sequence. The main, and initial, shared clinical phenotype among VRK1 pathogenic variants is a distal progressive loss of motor and/or sensory function, which includes diseases such as spinal muscular atrophy, Charcot-Marie-Tooth, amyotrophic lateral sclerosis and hereditary spastic paraplegia. In most cases, symptoms start early in infancy, or in utero, and are slowly progressive. Additional neurological symptoms vary among non-related patients, probably because of their different VRK1 variants and their genetic background. The underlying common pathogenic mechanism, by its functional impairment, is a likely consequence of the roles that the VRK1 protein plays in the regulation on the stability and assembly of Cajal bodies, which affect RNA maturation and processing, neuronal migration of RNPs along axons, and DNA-damage responses. Alterations of these processes are associated with several neuro sensory or motor syndromes. The clinical heterogeneity of the neurological phenotypes associated with VRK1 is a likely consequence of the protein complexes in which VRK1 is integrated, which include several proteins known to be associated with Cajal bodies and DNA damage responses. Several hereditary distal neurological diseases are a consequence of pathogenic variants in genes that alter these cellular functions. We conclude that VRK1-related distal hereditary neuropathies and motor neuron diseases represent a novel subgroup of Cajal body related neurological syndromes.

Cajal bodies: Evolutionarily conserved nuclear biomolecular condensates with properties unique to plants.

Proper orchestration of the thousands of biochemical processes that are essential to the life of every cell requires highly organized cellular compartmentalization of dedicated microenvironments. There are 2 ways to create this intracellular segregation to optimize cellular function. One way is to create specific organelles, enclosed spaces bounded by lipid membranes that regulate macromolecular flux in and out of the compartment. A second way is via membraneless biomolecular condensates that form due to to liquid-liquid phase separation. Although research on these membraneless condensates has historically been performed using animal and fungal systems, recent studies have explored basic principles governing the assembly, properties, and functions of membraneless compartments in plants. In this review, we discuss how phase separation is involved in a variety of key processes occurring in Cajal bodies (CBs), a type of biomolecular condensate found in nuclei. These processes include RNA metabolism, formation of ribonucleoproteins involved in transcription, RNA splicing, ribosome biogenesis, and telomere maintenance. Besides these primary roles of CBs, we discuss unique plant-specific functions of CBs in RNA-based regulatory pathways such as nonsense-mediated mRNA decay, mRNA retention, and RNA silencing. Finally, we summarize recent progress and discuss the functions of CBs in responses to pathogen attacks and abiotic stresses, responses that may be regulated via mechanisms governed by polyADP-ribosylation. Thus, plant CBs are emerging as highly complex and multifunctional biomolecular condensates that are involved in a surprisingly diverse range of molecular mechanisms that we are just beginning to appreciate.

Multiscale Modeling of Protein-RNA Condensation in and Out of Equilibrium.

A vast number of intracellular membraneless bodies also known as biomolecular condensates form through a liquid-liquid phase separation (LLPS) of biomolecules. To date, phase separation has been identified as the main driving force for a membraneless organelles such as nucleoli, Cajal bodies, stress granules, and chromatin compartments. Recently, the protein-RNA condensation is receiving increased attention, because it is closely related to the biological function of cells such as transcription, translation, and RNA metabolism. Despite the multidisciplinary efforts put forth to study the biophysical properties of protein-RNA condensates, there are many fundamental unanswered questions regarding the mechanism of formation and regulation of protein-RNA condensates in eukaryotic cells. Major challenges in studying protein-RNA condensation stem from (i) the molecular heterogeneity and conformational flexibility of RNA and protein chains and (ii) the nonequilibrium nature of transcription and cellular environment. Computer simulations, bioinformatics, and mathematical models are uniquely positioned for shedding light on the microscopic nature of protein-RNA phase separation. To this end, there is an urgent need for innovative models with the right spatiotemporal resolution for confronting the experimental observables in a comprehensive and physics-based manner. In this chapter, we will summarize the currently emerging research efforts, which employ atomistic and coarse-grained molecular models and field theoretical models to understand equilibrium and nonequilibrium aspects of protein-RNA condensation.

SUMO conjugation regulates the activity of the Integrator complex.

RNA polymerase II (RNAPII) transcribes small nuclear RNA (snRNA) genes in close proximity to Cajal bodies, subnuclear compartments that depend on the SUMO isopeptidase USPL1 for their assembly. We show here that overexpression of USPL1 as well as of another nuclear SUMO isopeptidase, SENP6, alters snRNA 3'-end cleavage, a process carried out by the Integrator complex. Beyond its role in snRNA biogenesis, this complex is responsible for regulating the expression of different RNAPII transcripts. While several subunits of the complex are SUMO conjugation substrates, we found that the SUMOylation of the INTS11 subunit is regulated by USPL1 and SENP6. We defined Lys381, Lys462 and Lys475 as bona fide SUMO attachment sites on INTS11 and observed that SUMOylation of this protein modulates its subcellular localization and is required for Integrator activity. Moreover, while an INTS11 SUMOylation-deficient mutant is still capable of interacting with INTS4 and INTS9, its interaction with other subunits of the complex is affected. These findings point to a regulatory role for SUMO conjugation on Integrator activity and suggest the involvement of INTS11 SUMOylation in the assembly of the complex. Furthermore, this work adds Integrator-dependent RNA processing to the growing list of cellular processes regulated by SUMO conjugation.

The coilin N-terminus mediates multivalent interactions between coilin and Nopp140 to form and maintain Cajal bodies.

Cajal bodies (CBs) are ubiquitous nuclear membraneless organelles (MLOs) that concentrate and promote efficient biogenesis of snRNA-protein complexes involved in splicing (snRNPs). Depletion of the CB scaffolding protein coilin disperses snRNPs, making CBs a model system for studying the structure and function of MLOs. Although it is assumed that CBs form through condensation, the biomolecular interactions responsible remain elusive. Here, we discover the unexpected capacity of coilin's N-terminal domain (NTD) to form extensive fibrils in the cytoplasm and discrete nuclear puncta in vivo. Single amino acid mutational analysis reveals distinct molecular interactions between coilin NTD proteins to form fibrils and additional NTD interactions with the nuclear Nopp140 protein to form puncta. We provide evidence that Nopp140 has condensation capacity and is required for CB assembly. From these observations, we propose a model in which coilin NTD-NTD mediated assemblies make multivalent contacts with Nopp140 to achieve biomolecular condensation in the nucleus.

Function of Cajal Bodies in Nuclear RNA Retention in A. thaliana Leaves Subjected to Hypoxia.

Retention of RNA in the nucleus precisely regulates the time and rate of translation and controls transcriptional bursts that can generate profound variability in mRNA levels among identical cells in tissues. In this study, we investigated the function of Cajal bodies (CBs) in RNA retention in A. thaliana leaf nuclei during hypoxia stress was investigated. It was observed that in ncb-1 mutants with a complete absence of CBs, the accumulation of poly(A+) RNA in the leaf nuclei was lower than that in wt under stress. Moreover, unlike in root cells, CBs store less RNA, and RNA retention in the nuclei is much less intense. Our results reveal that the function of CBs in the accumulation of RNA in nuclei under stress depends on the plant organ. Additionally, in ncb-1, retention of introns of mRNA RPB1 (largest subunit of RNA polymerase II) mRNA was observed. However, this isoform is highly accumulated in the nucleus. It thus follows that intron retention in transcripts is more important than CBs for the accumulation of RNA in nuclei. Accumulated mRNAs with introns in the nucleus could escape transcript degradation by NMD (nonsense-mediated mRNA decay). From non-fully spliced mRNAs in ncb-1 nuclei, whose levels increase during hypoxia, introns are removed during reoxygenation. Then, the mRNA is transferred to the cytoplasm, and the RPB1 protein is translated. Despite the accumulation of isoforms in nuclei with retention of introns in reoxygenation, ncb-1 coped much worse with long hypoxia, and manifested faster yellowing and shrinkage of leaves.

The 3' Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies' association with histone locus bodies.

Non-polyadenylated mRNAs of replication-dependent histones (RDHs) are synthesized by RNA polymerase II (Pol II) at histone locus bodies (HLBs). HLBs frequently associate with Cajal bodies (CBs), in which 3'-end processing factors for RDH genes are enriched; however, this association's role in transcription termination of RDH genes remains unclear. Here, we show that Pol II pauses immediately upstream of transcript end sites of RDH genes and Mediator plays a role in this Pol II pausing through CBs' association with HLBs. Disruption of the Mediator docking site for Little elongation complex (LEC)-Cap binding complex (CBC)-Negative elongation factor (NELF), components of CBs, interferes with CBs' association with HLBs and 3' Pol II pausing, resulting in increased aberrant unprocessed RDH gene transcripts. Our findings suggest Mediator's involvement in CBs' association with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing of RDH genes by supplying 3'-end processing factors.

Functional nuclear retention of pre-mRNA involving Cajal bodies during meiotic prophase in European larch (Larix decidua).

Gene regulation ensures that the appropriate genes are expressed at the proper time. Nuclear retention of incompletely spliced or mature mRNAs is emerging as a novel, previously underappreciated layer of posttranscriptional regulation. Studies on this phenomenon indicated that it exerts a significant influence on the regulation of gene expression by regulating export and translation delay, which allows the synthesis of specific proteins in response to a stimulus or at strictly controlled time points, for example, during cell differentiation or development. Here, we show that transcription in microsporocytes of European larch (Larix decidua) occurs in a pulsatile manner during prophase of the first meiotic division. Transcriptional activity was then silenced after each pulse. However, the transcripts synthesized were not exported immediately to the cytoplasm but were retained in the nucleoplasm and Cajal bodies (CBs). In contrast to the nucleoplasm, we did not detect mature transcripts in CBs, which only stored nonfully spliced transcripts with retained introns. Notably, the retained introns were spliced at precisely defined times, and fully mature mRNAs were released into the cytoplasm for translation. As similar processes have been observed during spermatogenesis in animals, our results illustrate an evolutionarily conserved mechanism of gene expression regulation during generative cells development in Eukaryota.

UHMK1-dependent phosphorylation of Cajal body protein coilin alters 5-FU sensitivity in colon cancer cells.

Resistance to 5-fluorouracil (5-FU) in chemotherapy and recurrence of colorectal tumors is a serious concern that impedes improvements to clinical outcomes. In the present study, we found that conditioned medium (CM) derived from 5-FU-resistant HCT-8/FU cells reduced 5-FU chemosensitivity in HCT-8 colon cancer cells, with corresponding changes to number and morphology of Cajal bodies (CBs) as observable nuclear structures. We found that U2AF homology motif kinase 1 (UHMK1) altered CB disassembly and reassembly and regulated the phosphorylation of coilin, a major component of CBs. This subsequently resulted in a large number of variations in RNA alternative splicing that affected cell survival following 5-FU treatment, induced changes in intracellular phenotype, and transmitted preadaptive signals to adjacent cells in the tumor microenvironment (TME). Our findings suggest that CBs may be useful for indicating drug sensitivity or resistance in tumor cells in response to stress signals. The results also suggest that UHMK1 may be an important factor for maintaining CB structure and morphology by regulating splicing events, especially following cellular exposure to cytotoxic drugs. Video Abstract.

RNA in formation and regulation of transcriptional condensates.

Macroscopic membraneless organelles containing RNA such as the nucleoli, germ granules, and the Cajal body have been known for decades. These biomolecular condensates are liquid-like bodies that can be formed by a phase transition. Recent evidence has revealed the presence of similar microscopic condensates associated with the transcription of genes. This brief article summarizes thoughts about the importance of condensates in the regulation of transcription and how RNA molecules, as components of such condensates, control the synthesis of RNA. Models and experimental data suggest that RNAs from enhancers facilitate the formation of a condensate that stabilizes the binding of transcription factors and accounts for a burst of transcription at the promoter. Termination of this burst is pictured as a nonequilibrium feedback loop where additional RNA destabilizes the condensate.

DHX15-independent roles for TFIP11 in U6 snRNA modification, U4/U6.U5 tri-snRNP assembly and pre-mRNA splicing fidelity.

The U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2'-O-methylation being most common. However, how U6 2'-O-methylation is regulated remains largely unknown. Here we report that TFIP11, the human homolog of the yeast spliceosome disassembly factor Ntr1, localizes to nucleoli and Cajal Bodies and is essential for the 2'-O-methylation of U6. Mechanistically, we demonstrate that TFIP11 knockdown reduces the association of U6 snRNA with fibrillarin and associated snoRNAs, therefore altering U6 2'-O-methylation. We show U6 snRNA hypomethylation is associated with changes in assembly of the U4/U6.U5 tri-snRNP leading to defects in spliceosome assembly and alterations in splicing fidelity. Strikingly, this function of TFIP11 is independent of the RNA helicase DHX15, its known partner in yeast. In sum, our study demonstrates an unrecognized function for TFIP11 in U6 snRNP modification and U4/U6.U5 tri-snRNP assembly, identifying TFIP11 as a critical spliceosome assembly regulator.

SnoRNA guide activities: real and ambiguous.

In eukaryotes, rRNAs and spliceosomal snRNAs are heavily modified post-transcriptionally. Pseudouridylation and 2'-O-methylation are the most abundant types of RNA modifications. They are mediated by modification guide RNAs, also known as small nucleolar (sno)RNAs and small Cajal body-specific (sca)RNAs. We used yeast and vertebrate cells to test guide activities predicted for a number of snoRNAs, based on their regions of complementarity with rRNAs. We showed that human SNORA24 is a genuine guide RNA for 18S-Ψ609, despite some noncanonical base-pairing with its target. At the same time, we found quite a few snoRNAs that have the ability to base-pair with rRNAs and can induce predicted modifications in artificial substrate RNAs, but do not modify the same target sequence within endogenous rRNA molecules. Furthermore, certain fragments of rRNAs can be modified by the endogenous yeast modification machinery when inserted into an artificial backbone RNA, even though the same sequences are not modified in endogenous yeast rRNAs. In Xenopus cells, a guide RNA generated from scaRNA, but not from snoRNA, could induce an additional pseudouridylation of U2 snRNA at position 60; both guide RNAs were equally active on a U2 snRNA-specific substrate in yeast cells. Thus, post-transcriptional modification of functionally important RNAs, such as rRNAs and snRNAs, is highly regulated and more complex than simply strong base-pairing between a guide RNA and substrate RNA. We discuss possible regulatory roles for these unexpected modifications.

DMA-tudor interaction modules control the specificity of in vivo condensates.

Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology.

The Cajal body protein coilin is a regulator of the miR-210 hypoxamiR and influences MIR210HG alternative splicing.

Hypoxia is a severe stressor to cellular homeostasis. At the cellular level, low oxygen triggers the transcription of a variety of genes supporting cell survival and oxygen homeostasis mediated by transcription factors, such as hypoxia-inducible factors (HIFs). Among many determinants dictating cell responses to hypoxia and HIFs are microRNAs (miRNAs). Cajal bodies (CBs), subnuclear structures involved in ribonucleoprotein biogenesis, have been recently proven to contribute to miRNA processing and biogenesis but have not been studied under hypoxia. Here, we show, for the first time, a hypoxia-dependent increase in CB number in WI-38 primary fibroblasts, which normally have very few CBs. Additionally, the CB marker protein coilin is upregulated in hypoxic WI-38 cells. However, the hypoxic coilin upregulation was not seen in transformed cell lines. Furthermore, we found that coilin is needed for the hypoxic induction of a well-known hypoxia-induced miRNA (hypoxamiR), miR-210, as well as for the hypoxia-induced alternative splicing of the miR-210 host gene, MIR210HG. These findings provide a new link in the physiological understanding of coilin, CBs and miRNA dysregulation in hypoxic pathology.

SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes.

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome-the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.

G-patch domain-containing protein 4 localizes to both the nucleoli and Cajal bodies and regulates cell growth and nucleolar structure.

Ribosome formation occurs in the nucleolus through interaction with various trans-acting factors. Therefore, hundreds of nucleolar proteins have a function in ribosome formation, although the precise function of each nucleolar protein in ribosome formation is largely unclear. We have previously identified an uncharacterized protein, G-patch domain-containing protein 4 (GPATCH4 or G4), as a component of the pre-ribosomes purified with either nucleolin (NCL) or NPM1. In this present study, we sought to clarify the localization and function of G4. We identified that G4 localizes to both the nucleolus and the Cajal body. Although knockdown of G4 did not have a significant effect on pre-ribosomal RNA processing, cell growth did decrease. Interestingly, G4 knockdown also decreased the number of fibrillar center and dense fibrillar component regions inside the nucleolus. This data has identified G4 as a novel nucleolar protein involved in the regulation of cell growth and nucleolar structure.