ABSTRACT
The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an 'assembly chaperone' by building up a stable precursor Sm protein ring structure. Here, we show that-apart from its autophagic role-the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.
Subject(s)
Autophagy-Related Protein-1 Homolog/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Ribonucleoproteins, Small Nuclear/biosynthesis , Autophagy-Related Protein-1 Homolog/antagonists & inhibitors , Autophagy-Related Protein-1 Homolog/physiology , Cell Line , Coiled Bodies , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/physiology , Ion Channels/metabolism , Phosphorylation , Protein-Arginine N-Methyltransferases/metabolismABSTRACT
The activity of the SMN complex in promoting the assembly of pre-mRNA processing UsnRNPs correlates with condensation of the complex in nuclear Cajal bodies. While mechanistic details of its activity have been elucidated, the molecular basis for condensation remains unclear. High SMN complex phosphorylation suggests extensive regulation. Here, we report on systematic siRNA-based screening for modulators of the capacity of SMN to condense in Cajal bodies and identify mTOR and ribosomal protein S6 kinase ß-1 as key regulators. Proteomic analysis reveals TOR-dependent phosphorylations in SMN complex subunits. Using stably expressed or optogenetically controlled phospho mutants, we demonstrate that serine 49 and 63 phosphorylation of human SMN controls the capacity of the complex to condense in Cajal bodies via liquid-liquid phase separation. Our findings link SMN complex condensation and UsnRNP biogenesis to cellular energy levels and suggest modulation of TOR signaling as a rational concept for therapy of the SMN-linked neuromuscular disorder spinal muscular atrophy.
Subject(s)
Ribonucleoproteins, Small Nuclear/biosynthesis , SMN Complex Proteins/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Cell Nucleus/metabolism , HeLa Cells , Humans , Mutation/genetics , Phosphorylation , Phosphoserine/metabolism , Protein Multimerization , Proteomics , Reproducibility of Results , Ribonucleoproteins, Small Nuclear/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolismABSTRACT
About 10% of cancer cells employ the "alternative lengthening of telomeres" (ALT) pathway instead of re-activating the hTERT subunit of human telomerase. The hTR RNA subunit is also abnormally silenced in some ALT+ cells not expressing hTERT, suggesting a possible negative non-canonical impact of hTR on ALT. Indeed, we show that ectopically expressed hTR reduces phosphorylation of ssDNA-binding protein RPA (p-RPAS33 ) at ALT telomeres by promoting the hnRNPA1- and DNA-PK-dependent depletion of RPA. The resulting defective ATR checkpoint signaling at telomeres impairs recruitment of the homologous recombination protein, RAD51. This induces ALT telomere fragility, increases POLD3-dependent C-circle production, and promotes the recruitment of the DNA damage marker 53BP1. In ALT+ cells that naturally retain hTR expression, NHP2 H/ACA ribonucleoprotein levels are downregulated, likely in order to restrain DNA damage response (DDR) activation at telomeres through reduced 53BP1 recruitment. This unexpected role of NHP2 is independent from hTR's non-canonical function in modulating telomeric p-RPAS33 . Collectively, our study shines new light on the interference between telomerase- and ALT-dependent pathways and unravels a crucial role for hTR and NHP2 in DDR regulation at ALT telomeres.
Subject(s)
Nuclear Proteins/biosynthesis , RNA/genetics , Ribonucleoproteins, Small Nuclear/biosynthesis , Telomerase/genetics , Telomere Homeostasis/physiology , Telomere/genetics , DNA Damage/genetics , DNA Repair/genetics , DNA-Activated Protein Kinase/metabolism , Down-Regulation , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Humans , Neoplasms/genetics , Rad51 Recombinase/metabolismABSTRACT
Ubiquitous deficiency in the survival motor neuron (SMN) protein causes death of motor neurons-a hallmark of the neurodegenerative disease spinal muscular atrophy (SMA)-through poorly understood mechanisms. Here, we show that the function of SMN in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) regulates alternative splicing of Mdm2 and Mdm4, two nonredundant repressors of p53. Decreased inclusion of critical Mdm2 and Mdm4 exons is most prominent in SMA motor neurons and correlates with both snRNP reduction and p53 activation in vivo. Importantly, increased skipping of Mdm2 and Mdm4 exons regulated by SMN is necessary and sufficient to synergistically elicit robust p53 activation in wild-type mice. Conversely, restoration of full-length Mdm2 and Mdm4 suppresses p53 induction and motor neuron degeneration in SMA mice. These findings reveal that loss of SMN-dependent regulation of Mdm2 and Mdm4 alternative splicing underlies p53-mediated death of motor neurons in SMA, establishing a causal link between snRNP dysfunction and neurodegeneration.
Subject(s)
Alternative Splicing , Motor Neurons/metabolism , Muscular Atrophy, Spinal/genetics , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins/genetics , Animals , Cell Death , Exons , Mice , Motor Neurons/pathology , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy, Spinal/physiopathology , NIH 3T3 Cells , Nerve Degeneration/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Ribonucleoproteins, Small Nuclear/biosynthesis , Tumor Suppressor Protein p53/metabolismABSTRACT
In cytoplasm, the survival of motor neuron (SMN) complex delivers pre-small nuclear RNAs (pre-snRNAs) to the heptameric Sm ring for the assembly of the ring complex on pre-snRNAs at the conserved Sm site [A(U)4-6G]. Gemin5, a WD40 protein component of the SMN complex, is responsible for recognizing pre-snRNAs. In addition, Gemin5 has been reported to specifically bind to the m7G cap. In this study, we show that the WD40 domain of Gemin5 is both necessary and sufficient for binding the Sm site of pre-snRNAs by isothermal titration calorimetry (ITC) and mutagenesis assays. We further determined the crystal structures of the WD40 domain of Gemin5 in complex with the Sm site or m7G cap of pre-snRNA, which reveal that the WD40 domain of Gemin5 recognizes the Sm site and m7G cap of pre-snRNAs via two distinct binding sites by respective base-specific interactions. In addition, we also uncovered a novel role of Gemin5 in escorting the truncated forms of U1 pre-snRNAs for proper disposal. Overall, the elucidated Gemin5 structures will contribute to a better understanding of Gemin5 in small nuclear ribonucleic protein (snRNP) biogenesis as well as, potentially, other cellular activities.
Subject(s)
Models, Molecular , RNA Precursors/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , SMN Complex Proteins/chemistry , SMN Complex Proteins/metabolism , Binding Sites , Cell Line , Crystallization , HEK293 Cells , Humans , Point Mutation , Protein Binding , Protein Domains/genetics , Protein Structure, Tertiary , Protein Transport , RNA Precursors/chemistry , Ribonucleoproteins, Small Nuclear/biosynthesis , SMN Complex Proteins/geneticsABSTRACT
The formation of macromolecular complexes within the crowded environment of cells often requires aid from assembly chaperones. PRMT5 and SMN complexes mediate this task for the assembly of the common core of pre-mRNA processing small nuclear ribonucleoprotein particles (snRNPs). Core formation is initiated by the PRMT5-complex subunit pICln, which pre-arranges the core proteins into spatial positions occupied in the assembled snRNP. The SMN complex then accepts these pICln-bound proteins and unites them with small nuclear RNA (snRNA). Here, we have analyzed how newly synthesized snRNP proteins are channeled into the assembly pathway to evade mis-assembly. We show that they initially remain bound to the ribosome near the polypeptide exit tunnel and dissociate upon association with pICln. Coincident with its release activity, pICln ensures the formation of cognate heterooligomers and their chaperoned guidance into the assembly pathway. Our study identifies the ribosomal quality control hub as a site where chaperone-mediated assembly of macromolecular complexes can be initiated.
Subject(s)
Ion Channels/metabolism , Ribonucleoproteins, Small Nuclear/biosynthesis , Ribosomes/metabolism , HEK293 Cells , Humans , Macromolecular Substances/metabolismABSTRACT
Small nuclear ribonucleoproteins (snRNPs) play a crucial role in pre-mRNA splicing in all eukaryotic cells. In contrast to the relatively broad knowledge on snRNP assembly within the nucleus, the spatial organization of the cytoplasmic stages of their maturation remains poorly understood. Nevertheless, sparse research indicates that, similar to the nuclear steps, the crucial processes of cytoplasmic snRNP assembly may also be strictly spatially regulated. In European larch microsporocytes, it was determined that the cytoplasmic assembly of snRNPs within a cell might occur in two distinct spatial manners, which depend on the rate of de novo snRNP formation in relation to the steady state of these particles within the nucleus. During periods of moderate expression of splicing elements, the cytoplasmic assembly of snRNPs occurred diffusely throughout the cytoplasm. Increased expression of both Sm proteins and U snRNA triggered the accumulation of these particles within distinct, non-membranous RNP-rich granules, which are referred to as snRNP-rich cytoplasmic bodies.
Subject(s)
Larix/metabolism , Ribonucleoproteins, Small Nuclear/biosynthesis , Cytoplasm/metabolism , RNA SplicingABSTRACT
Small nuclear ribonucleoproteins (snRNPs), which are required for pre-mRNA splicing, contain extensively modified snRNA. Small Cajal body-specific ribonucleoproteins (scaRNPs) mediate these modifications. It is unknown how the box C/D class of scaRNPs localizes to Cajal Bodies (CBs). The processing of box C/D scaRNA is also unclear. Here, we explore the processing of box C/D scaRNA 2 and 9 by coilin. We also broaden our investigation to include WRAP53 and SMN, which accumulate in CBs, play a role in RNP biogenesis and associate with coilin. These studies demonstrate that the processing of an ectopically expressed scaRNA2 is altered upon the reduction of coilin, WRAP53 or SMN, but the extent and direction of this change varies depending on the protein reduced. We also show that box C/D scaRNP activity is reduced in a cell line derived from coilin knockout mice. Collectively, the findings presented here further implicate coilin as being a direct participant in the formation of box C/D scaRNPs, and demonstrate that WRAP53 and SMN may also play a role, but the activity of these proteins is divergent to coilin.
Subject(s)
Coiled Bodies/metabolism , RNA Splicing/genetics , RNA, Small Nuclear/biosynthesis , Ribonucleoproteins, Small Nuclear/biosynthesis , Animals , Coiled Bodies/genetics , Cyclic AMP Response Element-Binding Protein/genetics , HeLa Cells , Humans , Mice , Mice, Knockout , Molecular Chaperones , RNA Precursors/genetics , RNA, Small Nuclear/genetics , Ribonucleoproteins, Small Nuclear/genetics , Telomerase/geneticsABSTRACT
By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis.
Subject(s)
Cyclin T/metabolism , Cyclin-Dependent Kinase 9/metabolism , Positive Transcriptional Elongation Factor B/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , Transcription, Genetic/physiology , Cyclin T/genetics , Cyclin-Dependent Kinase 9/genetics , HEK293 Cells , HeLa Cells , Humans , Methyltransferases/biosynthesis , Methyltransferases/genetics , Positive Transcriptional Elongation Factor B/genetics , Protein Biosynthesis/physiology , RNA-Binding Proteins/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Ribonucleoproteins/biosynthesis , Ribonucleoproteins/genetics , Ribonucleoproteins, Small Nuclear/biosynthesis , Ribonucleoproteins, Small Nuclear/genetics , Transcription Factors , Transcriptional Elongation FactorsABSTRACT
Cajal bodies (CBs) are subnuclear domains that participate in the biogenesis of small nuclear ribonucleoproteins (snRNPs) and telomerase. CBs are found in cells with high splicing demands, such as neuronal and cancer cells. The purpose of this review is to highlight what is known about the signals that impact the formation and activity of CBs. Particular attention is paid to phosphorylation as a major regulator of CB formation and composition, but a non-biochemical mediated pathway (mechanotransduction) that impacts CBs is also discussed. Amongst the CB components, recently published work on coilin (the CB marker protein) strongly suggests that this protein, and the CB by extension, is a global sensor that responds to environmental signals. Disruption of these signals, which would result in a decreased capacity to generate snRNPs and telomerase, is predicted to be beneficial in the treatment of cancer.
Subject(s)
Coiled Bodies/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , Animals , Humans , Nuclear Proteins/metabolism , Phosphorylation , Ribonucleoproteins, Small Nuclear/biosynthesis , Telomerase/biosynthesis , Telomerase/metabolismABSTRACT
The cell nucleus contains dozens of subcompartments that separate biochemical processes into confined spaces. Cajal bodies (CBs) were discovered more than 100 years ago, but only extensive research in the past decades revealed the surprising complexity of molecular and cellular functions taking place in these structures. Many protein and RNA species are modified and assembled within CBs, which have emerged as a meeting place and factory for ribonucleoprotein (RNP) particles involved in splicing, ribosome biogenesis and telomere maintenance. Recently, a distinct structure near histone gene clusters--the Histone locus body (HLB)--was discovered. Involved in histone mRNA 3'-end formation, HLBs can share several components with CBs. Whether the appearance of distinct HLBs is simply a matter of altered affinity between these structures or of an alternate mode of CB assembly is unknown. However, both structures share basic assembly properties, in which transcription plays a decisive role in initiation. After this seeding event, additional components associate in random order. This appears to be a widespread mechanism for body assembly. CB assembly encompasses an additional layer of complexity, whereby a set of pre-existing substructures can be integrated into mature CBs. We propose this as a multi-seeding model of CB assembly.
Subject(s)
Cell Nucleus/metabolism , Coiled Bodies/genetics , Coiled Bodies/metabolism , Animals , Histones/genetics , Humans , RNA/genetics , RNA/metabolism , RNA Splicing , Ribonucleoproteins, Small Nuclear/biosynthesis , Ribonucleoproteins, Small Nuclear/metabolism , Ribonucleoproteins, Small Nucleolar/biosynthesis , Ribonucleoproteins, Small Nucleolar/metabolismABSTRACT
The AAA+ ATPases pontin and reptin function in a staggering array of cellular processes including chromatin remodeling, transcriptional regulation, DNA damage repair, and assembly of macromolecular complexes, such as RNA polymerase II and small nucleolar (sno) RNPs. However, the molecular mechanism for all of these AAA+ ATPase associated activities is unknown. Here we document that, during the biogenesis of H/ACA RNPs (including telomerase), the assembly factor SHQ1 holds the pseudouridine synthase NAP57/dyskerin in a viselike grip, and that pontin and reptin (as components of the R2TP complex) are required to pry NAP57 from SHQ1. Significantly, the NAP57 domain captured by SHQ1 harbors most mutations underlying X-linked dyskeratosis congenita (X-DC) implicating the interface between the two proteins as a target of this bone marrow failure syndrome. Homing in on the essential first steps of H/ACA RNP biogenesis, our findings provide the first insight into the mechanism of action of pontin and reptin in the assembly of macromolecular complexes.
Subject(s)
Carrier Proteins/physiology , DNA Helicases/physiology , Macromolecular Substances/metabolism , Ribonucleoproteins, Small Nuclear/biosynthesis , Ribonucleoproteins, Small Nucleolar/metabolism , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/physiology , Animals , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cells, Cultured , DNA Helicases/antagonists & inhibitors , DNA Helicases/genetics , DNA Helicases/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , HeLa Cells , Humans , Mice , Models, Biological , Protein Multimerization/genetics , Protein Multimerization/physiology , RNA, Small Interfering/pharmacology , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
The spinal muscular atrophy (SMA) protein, survival motor neuron (SMN), functions in the biogenesis of small nuclear ribonucleoproteins (snRNPs). SMN has also been implicated in tissue-specific functions; however, it remains unclear which of these is important for the etiology of SMA. Smn null mutants display larval lethality and show significant locomotion defects as well as reductions in minor-class spliceosomal snRNAs. Despite these reductions, we found no appreciable defects in the splicing of mRNAs containing minor-class introns. Transgenic expression of low levels of either wild-type or an SMA patient-derived form of SMN rescued the larval lethality and locomotor defects; however, snRNA levels were not restored. Thus, the snRNP biogenesis function of SMN is not a major contributor to the phenotype of Smn null mutants. These findings have major implications for SMA etiology because they show that SMN's role in snRNP biogenesis can be uncoupled from the organismal viability and locomotor defects.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Locomotion , Muscular Atrophy, Spinal/pathology , Muscular Atrophy, Spinal/physiopathology , RNA-Binding Proteins/metabolism , Ribonucleoproteins, Small Nuclear/biosynthesis , Animals , Animals, Genetically Modified , Base Sequence , Cell Survival , Disease Models, Animal , Drosophila Proteins/chemistry , Drosophila Proteins/deficiency , Humans , Introns/genetics , Larva , Molecular Sequence Data , Mutation/genetics , Protein Structure, Quaternary , RNA Splicing/genetics , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , RNA, Small Nuclear/metabolism , RNA-Binding Proteins/chemistry , Real-Time Polymerase Chain Reaction , Ribonucleoproteins, Small Nuclear/metabolismABSTRACT
Coilin is known as the marker protein for Cajal bodies (CBs), subnuclear domains important for the biogenesis of small nuclear ribonucleoproteins (snRNPs) which function in pre-mRNA splicing. CBs associate non-randomly with U1 and U2 gene loci, which produce the small nuclear RNA (snRNA) component of the respective snRNP. Despite recognition as the CB marker protein, coilin is primarily nucleoplasmic, and the function of this fraction is not fully characterized. Here we show that coilin binds double stranded DNA and has RNase activity in vitro. U1 and U2 snRNAs undergo a processing event of the primary transcript prior to incorporation in the snRNP. We find that coilin displays RNase activity within the CU region of the U2 snRNA primary transcript in vitro, and that coilin knockdown results in accumulation of the 3' pre-processed U1 and U2 snRNA. These findings present new characteristics of coilin in vitro, and suggest additional functions of the protein in vivo.
Subject(s)
DNA/metabolism , Nuclear Proteins/metabolism , RNA Processing, Post-Transcriptional , RNA, Small Nuclear/metabolism , Ribonucleases/metabolism , Cell Nucleus/metabolism , Gene Knockdown Techniques , Genetic Loci/genetics , HeLa Cells , Humans , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Protein Binding , RNA Precursors/metabolism , Ribonucleoproteins, Small Nuclear/biosynthesisABSTRACT
The SMN protein is essential and participates in the assembly of macromolecular complexes of RNA and protein in all cells. The best-characterized function of SMN is as an assembler of spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN performs this function as part of a complex with several other proteins called Gemins. snRNPs are assembled in the cytoplasm in a stepwise manner and then are imported to the nucleus where they participate globally in the splicing of pre-mRNA. Mutations in the SMN1 gene result in the motor neuron disease, spinal muscular atrophy (SMA). Most of these mutations result in a reduction in the expression levels of the SMN protein, which, in turn, results in a reduction in snRNP assembly capacity. This review highlights current studies that have investigated the mechanism of SMN-dependent snRNP assembly, as well as the downstream effects on pre-mRNA splicing that result from a decrease in SMN. This article is part of a Special Issue entitled "RNA-Binding Proteins".
Subject(s)
Motor Neuron Disease/genetics , Motor Neurons/physiology , Muscular Atrophy, Spinal/genetics , Ribonucleoproteins, Small Nuclear/biosynthesis , Animals , Humans , Motor Neuron Disease/physiopathology , Muscular Atrophy, Spinal/physiopathology , Mutation/genetics , Mutation/physiology , Ribonucleoproteins, Small Nuclear/genetics , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 1 Protein/physiologyABSTRACT
Ribonucleoprotein (RNP) complexes function in nearly every facet of cellular activity. The spliceosome is an essential RNP that accurately identifies introns and catalytically removes the intervening sequences, providing exquisite control of spatial, temporal, and developmental gene expressions. U-snRNPs are the building blocks for the spliceosome. A significant amount of insight into the molecular assembly of these essential particles has recently come from a seemingly unexpected area of research: neurodegeneration. Survival motor neuron (SMN) performs an essential role in the maturation of snRNPs, while the homozygous loss of SMN1 results in the development of spinal muscular atrophy (SMA), a devastating neurodegenerative disease. In this review, the function of SMN is examined within the context of snRNP biogenesis and evidence is examined which suggests that the SMN functional defects in snRNP biogenesis may account for the motor neuron pathology observed in SMA.
Subject(s)
Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/metabolism , Ribonucleoproteins, Small Nuclear/biosynthesis , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 1 Protein/metabolism , Alternative Splicing , Axons/metabolism , Humans , Models, Neurological , Muscular Atrophy, Spinal/classification , Muscular Atrophy, Spinal/etiology , Protein Structure, Tertiary , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing , Survival of Motor Neuron 1 Protein/chemistry , Survival of Motor Neuron 2 Protein/genetics , Survival of Motor Neuron 2 Protein/metabolismABSTRACT
Virtually, all eukaryotic mRNAs are synthesized as precursor molecules that need to be extensively processed in order to serve as a blueprint for proteins. The three most prevalent processing steps are the capping reaction at the 5'-end, the removal of intervening sequences by splicing, and the formation of poly (A)-tails at the 3'-end of the message by polyadenylation. A large number of proteins and small nuclear ribonucleoprotein complexes (snRNPs) interact with the mRNA and enable the different maturation steps. This chapter focuses on the biogenesis of snRNPs, the major components of the pre-mRNA splicing machinery (spliceosome). A large body of evidence has revealed an intricate and segmented pathway for the formation of snRNPs that involves nucleo-cytoplasmic transport events and elaborates assembly strategies. We summarize the knowledge about the different steps with an emphasis on trans-acting factors of snRNP maturation of higher eukaryotes. WIREs RNA 2011 2 718-731 DOI: 10.1002/wrna.87 For further resources related to this article, please visit the WIREs website.
Subject(s)
Ribonucleoproteins, Small Nuclear/biosynthesis , Spliceosomes/metabolism , Active Transport, Cell Nucleus , Animals , Humans , Mice , Models, Biological , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/metabolism , RNA Polymerase II/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing , RNA, Small Nuclear/genetics , RNA, Small Nuclear/metabolism , Ribonucleoproteins, Small Nuclear/genetics , Spliceosomes/genetics , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 1 Protein/metabolismABSTRACT
Box H/ACA RNAs represent an abundant, evolutionarily conserved class of small noncoding RNAs. All H/ACA RNAs associate with a common set of proteins, and they function as ribonucleoprotein (RNP) enzymes mainly in the site-specific pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Some H/ACA RNPs function in the nucleolytic processing of precursor rRNA (pre-rRNA) and synthesis of telomeric DNA. Thus, H/ACA RNPs are essential for three fundamental cellular processes: protein synthesis, mRNA splicing, and maintenance of genome integrity. Recently, great progress has been made toward understanding of the biogenesis, intracellular trafficking, structure, and function of H/ACA RNPs.
Subject(s)
Coiled Bodies/metabolism , RNA, Untranslated/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , Active Transport, Cell Nucleus , Animals , Archaeal Proteins/metabolism , Evolution, Molecular , Genomic Instability , Humans , Intramolecular Transferases/metabolism , Models, Molecular , Nucleic Acid Conformation , Protein Biosynthesis , Protein Conformation , Pseudouridine/metabolism , RNA Processing, Post-Transcriptional , RNA Splicing , RNA, Messenger/metabolism , RNA, Ribosomal/metabolism , RNA, Untranslated/chemistry , Ribonucleoproteins, Small Nuclear/biosynthesis , Ribonucleoproteins, Small Nuclear/chemistry , Telomerase/metabolismSubject(s)
Gene Expression/genetics , Muscular Atrophy, Spinal/genetics , RNA Splicing , Ribonucleoproteins, Small Nuclear/biosynthesis , Spliceosomes/genetics , Active Transport, Cell Nucleus , Animals , Cytoplasm/metabolism , Humans , Protein Transport , RNA, Messenger/genetics , SMN Complex Proteins/physiology , Transcription, GeneticABSTRACT
H/ACA ribonucleoprotein particles are essential for ribosomal RNA and telomerase RNA processing and metabolism. Shq1p has been identified as an essential eukaryotic H/ACA small nucleolar (sno) ribonucleoparticle (snoRNP) biogenesis and assembly factor. Shq1p is postulated to be involved in the early biogenesis steps of H/ACA snoRNP complexes, and Shq1p depletion leads to a specific decrease in H/ACA small nucleolar RNA levels and to defects in ribosomal RNA processing. Shq1p contains two predicted domains as follows: an N-terminal CS (named after CHORD-containing proteins and SGT1) or HSP20-like domain, and a C-terminal region of high sequence homology called the Shq1 domain. Here we report the crystal structure and functional studies of the Saccharomyces cerevisiae Shq1p CS domain. The structure consists of a compact anti-parallel beta-sandwich fold that is composed of two beta-sheets containing four and three beta-strands, respectively, and a short alpha-helix. Deletion studies showed that the CS domain is required for the essential functions of Shq1p. Point mutations in residues Phe-6, Gln-10, and Lys-80 destabilize Shq1p in vivo and induce a temperature-sensitive phenotype with depletion of H/ACA small nucleolar RNAs and defects in rRNA processing. Although CS domains are frequently found in co-chaperones of the Hsp90 molecular chaperone, no interaction was detected between the Shq1p CS domain and yeast Hsp90 in vitro. These results show that the CS domain is essential for Shq1p function in H/ACA snoRNP biogenesis in vivo, possibly in an Hsp90-independent manner.