The Methylosome, a 20S Complex Containing JBP1 and pICln, Produces Dimethylarginine-Modified Sm Proteins

ABSTRACT snRNPs, integral components of the pre-mRNA splicing machinery, consist of seven Sm proteins which assemble in the cytoplasm as a ring structure on the snRNAs U1, U2, U4, and U5. The survival motor neuron (SMN) protein, the spinal muscular atrophy disease gene product, is crucial for snRNP core particle assembly in vivo. SMN binds preferentially and directly to the symmetrical dimethylarginine (sDMA)-modified arginine- and glycine-rich (RG-rich) domains of SmD1 and SmD3. We found that the unmodified, but not the sDMA-modified, RG domains of SmD1 and SmD3 associate with a 20S methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 and a JBP1-interacting protein, pICln. JBP1 binds SmD1 and SmD3 via their RG domains, while pICln binds the Sm domains. JBP1 produces sDMAs in the RG domain-containing Sm proteins. We further demonstrate the existence of a 6S complex that contains pICln, SmD1, and SmD3 but not JBP1. SmD3 from the methylosome, but not that from the 6S complex, can be transferred to the SMN complex in vitro. Together with previous results, these data indicate that methylation of Sm proteins by the methylosome directs Sm proteins to the SMN complex for assembly into snRNP core particles and suggest that the methylosome can regulate snRNP assembly.

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Negative Cooperativity between Gemin2 and RNA provides Insights into RNA Selection and the SMN Complex’s Release in snRNP Assembly

10.1101/312124 ◽

2018 ◽

Author(s):

Hongfei Yi ◽

Li Mu ◽

Congcong Shen ◽

Xi Kong ◽

Yingzhi Wang ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Muscular Atrophy ◽

Basic Mechanism ◽

Negative Cooperativity ◽

Sm Proteins ◽

Early Step ◽

Smn Complex ◽

Narrow State ◽

Snrnp Core

ABSTRACTThe 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.

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

Nucleic Acids Research ◽

10.1093/nar/gkz1135 ◽

2019 ◽

Vol 48 (2) ◽

pp. 895-911 ◽

Cited By ~ 1

Author(s):

Hongfei Yi ◽

Li Mu ◽

Congcong Shen ◽

Xi Kong ◽

Yingzhi Wang ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Muscular Atrophy ◽

Basic Mechanism ◽

Negative Cooperativity ◽

Sm Proteins ◽

Early Step ◽

Smn Complex ◽

Narrow State ◽

Snrnp Core

Abstract 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.

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Dephosphorylation of survival motor neurons (SMN) by PPM1G/PP2Cγ governs Cajal body localization and stability of the SMN complex

The Journal of Cell Biology ◽

10.1083/jcb.200704163 ◽

2007 ◽

Vol 179 (3) ◽

pp. 451-465 ◽

Cited By ~ 33

Author(s):

Sebastian Petri ◽

Matthias Grimmler ◽

Sabine Over ◽

Utz Fischer ◽

Oliver J. Gruss

Keyword(s):

Motor Neurons ◽

Survival Motor Neuron ◽

Cajal Body ◽

Sm Proteins ◽

Interacting Protein ◽

Survival Motor Neurons ◽

Small Nuclear Ribonucleoprotein ◽

Smn Complex ◽

Catalytically Active ◽

And Function

The survival motor neuron (SMN) complex functions in maturation of uridine-rich small nuclear ribonucleoprotein (RNP) particles. SMN mediates the cytoplasmic assembly of Sm proteins onto uridine-rich small RNAs, and then participates in targeting RNPs to nuclear Cajal bodies (CBs). Recent studies have suggested that phosphorylation might control localization and function of the SMN complex. Here, we show that the nuclear phosphatase PPM1G/PP2Cγ interacts with and dephosphorylates the SMN complex. Small interfering RNA knockdown of PPM1G leads to an altered phosphorylation pattern of SMN and Gemin3, loss of SMN from CBs, and reduced stability of SMN. Accumulation in CBs is restored upon overexpression of catalytically active, but not that of inactive, PPM1G. This demonstrates that PPM1G's phosphatase activity is necessary to maintain SMN subcellular distribution. Concomitant knockdown of unr interacting protein (unrip), a component implicated in cytoplasmic retention of the SMN complex, also rescues the localization defects. Our data suggest that an interplay between PPM1G and unrip determine compartment-specific phosphorylation patterns, localization, and function of the SMN complex.

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Composition of the Survival Motor Neuron (SMN) complex in Drosophila melanogaster

10.1101/468223 ◽

2018 ◽

Author(s):

A. Gregory Matera ◽

Amanda C. Raimer ◽

Casey A. Schmidt ◽

Jo A. Kelly ◽

Gaith N. Droby ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Motor Neuron ◽

Muscular Atrophy ◽

Structural Similarity ◽

Survival Motor Neuron ◽

Sm Proteins ◽

Native Promoter ◽

Smn Complex ◽

Biological Studies ◽

Smn Protein

AbstractSpinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated the composition of the SMN complex in Drosophila melanogaster. Using a stable transgenic line that exclusively expresses Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

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Effects of Survival Motor Neuron Protein on Germ Cell Development in Mouse and Human

International Journal of Molecular Sciences ◽

10.3390/ijms22020661 ◽

2021 ◽

Vol 22 (2) ◽

pp. 661

Author(s):

Wei-Fang Chang ◽

Min Peng ◽

Jing Hsu ◽

Jie Xu ◽

Huan-Chieh Cho ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Motor Neuron ◽

Cell Biology ◽

Muscular Atrophy ◽

Primordial Germ Cell ◽

Cell Types ◽

Survival Motor Neuron

Survival motor neuron (SMN) is ubiquitously expressed in many cell types and its encoding gene, survival motor neuron 1 gene (SMN1), is highly conserved in various species. SMN is involved in the assembly of RNA spliceosomes, which are important for pre-mRNA splicing. A severe neurogenic disease, spinal muscular atrophy (SMA), is caused by the loss or mutation of SMN1 that specifically occurred in humans. We previously reported that SMN plays roles in stem cell biology in addition to its roles in neuron development. In this study, we investigated whether SMN can improve the propagation of spermatogonia stem cells (SSCs) and facilitate the spermatogenesis process. In in vitro culture, SSCs obtained from SMA model mice showed decreased growth rate accompanied by significantly reduced expression of spermatogonia marker promyelocytic leukemia zinc finger (PLZF) compared to those from heterozygous and wild-type littermates; whereas SMN overexpressed SSCs showed enhanced cell proliferation and improved potency. In vivo, the superior ability of homing and complete performance in differentiating progeny was shown in SMN overexpressed SSCs in host seminiferous tubule of transplant experiments compared to control groups. To gain insights into the roles of SMN in clinical infertility, we derived human induced pluripotent stem cells (hiPSCs) from azoospermia patients (AZ-hiPSCs) and from healthy control (ct-hiPSCs). Despite the otherwise comparable levels of hallmark iPCS markers, lower expression level of SMN1 was found in AZ-hiPSCs compared with control hiPSCs during in vitro primordial germ cell like cells (PGCLCs) differentiation. On the other hand, overexpressing hSMN1 in AZ-hiPSCs led to increased level of pluripotent markers such as OCT4 and KLF4 during PGCLC differentiation. Our work reveal novel roles of SMN in mammalian spermatogenesis and suggest new therapeutic targets for azoospermia treatment.

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Calpain system is altered in survival motor neuron-reduced cells from in vitro and in vivo spinal muscular atrophy models

Cell Death and Disease ◽

10.1038/s41419-020-2688-5 ◽

2020 ◽

Vol 11 (6) ◽

Cited By ~ 1

Author(s):

Sandra de la Fuente ◽

Alba Sansa ◽

Iván Hidalgo ◽

Nuria Vivancos ◽

Ricardo Romero-Guevara ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Calpain System

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A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II

The Journal of Cell Biology ◽

10.1083/jcb.152.1.75 ◽

2001 ◽

Vol 152 (1) ◽

pp. 75-86 ◽

Cited By ~ 154

Author(s):

Livio Pellizzoni ◽

Bernard Charroux ◽

Juri Rappsilber ◽

Matthias Mann ◽

Gideon Dreyfuss

Keyword(s):

Rna Polymerase ◽

Motor Neuron ◽

Rna Polymerase Ii ◽

Rna Helicase ◽

Survival Motor Neuron ◽

Dominant Negative Mutant ◽

Pol Ii ◽

Smn Complex

The survival motor neuron (SMN) protein, the protein product of the spinal muscular atrophy (SMA) disease gene, plays a role in the assembly and regeneration of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. By nanoelectrospray mass spectrometry, we identified RNA helicase A (RHA) as an SMN complex–associated protein. RHA is a DEAH box RNA helicase which binds RNA polymerase II (pol II) and reportedly functions in transcription. SMN interacts with RHA in vitro, and this interaction is impaired in mutant SMNs found in SMA patients. Coimmunoprecipitation demonstrated that the SMN complex is associated with pol II, snRNPs, and RHA in vivo. In vitro experiments suggest that RHA mediates the association of SMN with the COOH-terminal domain of pol II. Moreover, transfection of cells with a dominant negative mutant of SMN, SMNΔN27, causes accumulation of pol II, snRNPs, and RHA in nuclear structures that contain the known markers of gems and coiled bodies, and inhibits RNA pol I and pol II transcription in vivo. These findings indicate a functional as well as physical association of the SMN complex with pol II and suggest a role for the SMN complex in the assembly of the pol II transcription/processing machinery.

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

The Journal of Cell Biology ◽

10.1083/jcb.201611108 ◽

2017 ◽

Vol 216 (8) ◽

pp. 2391-2407 ◽

Cited By ~ 10

Author(s):

Archana Bairavasundaram Prusty ◽

Rajyalakshmi Meduri ◽

Bhupesh Kumar Prusty ◽

Jens Vanselow ◽

Andreas Schlosser ◽

...

Keyword(s):

Survival Motor Neuron ◽

Sm Proteins ◽

Down Regulation ◽

Small Nuclear Ribonucleoprotein ◽

Smn Complex ◽

Assembly Factors ◽

Nuclear Ribonucleoprotein ◽

Contrast Reduction ◽

Sm Protein

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

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Gemin3 Is an Essential Gene Required for Larval Motor Function and Pupation in Drosophila

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0024 ◽

2009 ◽

Vol 20 (1) ◽

pp. 90-101 ◽

Cited By ~ 37

Author(s):

Karl B. Shpargel ◽

Kavita Praveen ◽

T. K. Rajendra ◽

A. Gregory Matera

Keyword(s):

Protein Function ◽

Muscular Atrophy ◽

Essential Gene ◽

Survival Motor Neuron ◽

Loss Of Function ◽

Transposon Insertion ◽

Associated Proteins ◽

Smn Protein

The assembly of metazoan Sm-class small nuclear ribonucleoproteins (snRNPs) is an elaborate, step-wise process that takes place in multiple subcellular compartments. The initial steps, including formation of the core RNP, are mediated by the survival motor neuron (SMN) protein complex. Loss-of-function mutations in human SMN1 result in a neuromuscular disease called spinal muscular atrophy. The SMN complex is comprised of SMN and a number of tightly associated proteins, collectively called Gemins. In this report, we identify and characterize the fruitfly ortholog of the DEAD box protein, Gemin3. Drosophila Gemin3 (dGem3) colocalizes and interacts with dSMN in vitro and in vivo. RNA interference for dGem3 codepletes dSMN and inhibits efficient Sm core assembly in vitro. Transposon insertion mutations in Gemin3 are larval lethals and also codeplete dSMN. Transgenic overexpression of dGem3 rescues lethality, but overexpression of dSMN does not, indicating that loss of dSMN is not the primary cause of death. Gemin3 mutant larvae exhibit motor defects similar to previously characterized Smn alleles. Remarkably, appreciable numbers of Gemin3 mutants (along with one previously undescribed Smn allele) survive as larvae for several weeks without pupating. Our results demonstrate the conservation of Gemin3 protein function in metazoan snRNP assembly and reveal that loss of either Smn or Gemin3 can contribute to neuromuscular dysfunction.

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