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

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.

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

2019 ◽  
Vol 48 (2) ◽  
pp. 895-911 ◽  
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.

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

2001 ◽  
Vol 21 (24) ◽  
pp. 8289-8300 ◽  
Author(s):  
Westley J. Friesen ◽  
Sergey Paushkin ◽  
Anastasia Wyce ◽  
Severine Massenet ◽  
G. Scott Pesiridis ◽  
...  
Keyword(s):  
Muscular Atrophy ◽  
Ring Structure ◽  
Survival Motor Neuron ◽  
Sm Proteins ◽  
Particle Assembly ◽  
Interacting Protein ◽  
Smn Complex ◽  
Snrnp Core

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.

scholarly journals Structural Insights into Negative Cooperativity between Gemin2 and RNA in Sm-class snRNP Assembly

2019 ◽  
Author(s):  
Rundong Zhang
Keyword(s):  
Spinal Muscular Atrophy ◽  
Neurodegenerative Disease ◽  
Muscular Atrophy ◽  
Structural Mechanics ◽  
Atomic Resolution ◽  
Negative Cooperativity ◽  
Stem Loop ◽  
Smn Complex ◽  
Resolution Level ◽  
Structural Insights

ABSTRACTSm-class ribonucleoprotein particles (RNPs) are ring-shaped structures (Sm cores) formed by Sm hetero-heptamer around a segment of RNA, containing a nonameric oligoribonucleotide, PuAUUUNUGPu, followed by a stem-loop, and are basic structural modules critical for stability and functions of spliceosomal, telomerase and U7 RNPs. In the chaperones-assisted Sm core assembly, Gemin2 of the SMN complex, not only binds SmD1/D2/F/E/G (5Sm), but also serves as a checkpoint via a negative cooperativity mechanism uncovered in our recent study: Gemin2 constricts the horseshoe-shaped 5Sm in a narrow conformation from outside, preventing non-cognate RNA and SmD3/B from joining; only cognate RNA can bind inside 5Sm and widen 5Sm, dissociating Gemin2 from 5Sm and recruiting SmD3/B. However, the structural mechanics is unknown. Here I describe a coordinate-improved structure of 5Sm bound by Gemin2/SMN. Moreover, via new analysis, comparison of this structure with those of newly coordinate-improved Sm cores reveals the negative cooperativity mechanism between Gemin2 and RNA in binding 5Sm at atomic resolution level and provides structural insights into RNA selection and Gemin2’s release in Sm core assembly. Finally, implications in the evolution of the Sm-core assembly chaperoning machinery and the neurodegenerative disease spinal muscular atrophy caused by SMN deficiency are discussed.

scholarly journals The Survival of Motor Neurons Protein Determines the Capacity for snRNP Assembly: Biochemical Deficiency in Spinal Muscular Atrophy

2005 ◽  
Vol 25 (13) ◽  
pp. 5543-5551 ◽  
Author(s):  
Lili Wan ◽  
Daniel J. Battle ◽  
Jeongsik Yong ◽  
Amelie K. Gubitz ◽  
Stephen J. Kolb ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neurons ◽  
Muscular Atrophy ◽  
Protein Levels ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Smn Complex ◽  
Survival Of Motor Neurons ◽  
Smn Protein ◽  
Nuclear Ribonucleoprotein

ABSTRACT Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN, together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels, as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known. To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts. Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a biochemical activity in cells from patients with spinal muscular atrophy.

scholarly journals Chondrolectin affects cell survival and neuronal outgrowth in in vitro and in vivo models of spinal muscular atrophy

2013 ◽  
Vol 23 (4) ◽  
pp. 855-869 ◽  
Author(s):  
James N. Sleigh ◽  
Antón Barreiro-Iglesias ◽  
Peter L. Oliver ◽  
Angeliki Biba ◽  
Thomas Becker ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Cell Survival ◽  
Muscular Atrophy ◽  
In Vivo Models ◽  
Neuronal Outgrowth

scholarly journals Synthesis and Characterization of Pseudocantharidins, Novel Phosphatase Modulators That Promote the Inclusion of Exon 7 into the SMN (Survival of Motoneuron) pre-mRNA

2011 ◽  
Vol 286 (12) ◽  
pp. 10126-10136 ◽  
Author(s):  
Zhaiyi Zhang ◽  
Olga Kelemen ◽  
Maria A. van Santen ◽  
Sharon M. Yelton ◽  
Alison E. Wendlandt ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Central Element ◽  
Detectable Effect ◽  
Splice Site Selection ◽  
Eukaryotic Gene ◽  
Constitutive Splicing ◽  
New Compounds

Alternative pre-mRNA splicing is a central element of eukaryotic gene expression. Its deregulation can lead to disease, and methods to change splice site selection are developed as potential therapies. Spinal muscular atrophy is caused by the loss of the SMN1 (survival of motoneuron 1) gene. A therapeutic avenue for spinal muscular atrophy treatment is to promote exon 7 inclusion of the almost identical SMN2 (survival of motoneuron 2) gene. The splicing factor tra2-beta1 promotes inclusion of this exon and is antagonized by protein phosphatase (PP) 1. To identify new compounds that promote exon 7 inclusion, we synthesized analogs of cantharidin, an inhibitor of PP1, and PP2A. Three classes of compounds emerged from these studies. The first class blocks PP1 and PP2A activity, blocks constitutive splicing in vitro, and promotes exon 7 inclusion in vivo. The second class has no measurable effect on PP1 activity but activates PP2A. This class represents the first compounds described with these properties. These compounds cause a dephosphorylation of Thr-33 of tra2-beta1, which promotes exon 7 inclusion. The third class had no detectable effect on phosphatase activity and could promote exon 7 via allosteric effects. Our data show that subtle changes in similar compounds can turn a phosphatase inhibitor into an activator. These chemically related compounds influence alternative splicing by distinct mechanisms.

scholarly journals Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1636 ◽  
Author(s):  
Chris D. Balak ◽  
Jesse M. Hunter ◽  
Mary E. Ahearn ◽  
David Wiley ◽  
Gennaro D'urso ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Diagnostic Assays ◽  
Missense Variants ◽  
Thioester Bond ◽  
In The Wild ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

Background: X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 (UBA1). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the UBA1 gene, which contains the active adenylation domain (AAD). Methods: In this study, our group characterized the three known missense variants in vitro. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions in vitro to determine possible biochemical effects of the missense variants. Results: Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. Conclusions: Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1’s enzymatic activity with Ubiquitin, to other roles such as altering UBA1 mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important in vivo binding partners.  These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations’ biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.

scholarly journals In Vivo Translatome Profiling in Spinal Muscular Atrophy Reveals a Role for SMN Protein in Ribosome Biology

Cell Reports ◽  
2017 ◽  
Vol 21 (4) ◽  
pp. 953-965 ◽  
Author(s):  
Paola Bernabò ◽  
Toma Tebaldi ◽  
Ewout J.N. Groen ◽  
Fiona M. Lane ◽  
Elena Perenthaler ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Smn Protein

scholarly journals A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle

2007 ◽  
Vol 176 (6) ◽  
pp. 831-841 ◽  
Author(s):  
T.K. Rajendra ◽  
Graydon B. Gonsalvez ◽  
Michael P. Walker ◽  
Karl B. Shpargel ◽  
Helen K. Salz ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Spinal Muscular Atrophy ◽  
Motor Neurons ◽  
Striated Muscle ◽  
Flight Muscle ◽  
Muscular Atrophy ◽  
Survival Motor Neurons ◽  
Protein Levels

Mutations in human survival motor neurons 1 (SMN1) cause spinal muscular atrophy (SMA) and are associated with defects in assembly of small nuclear ribonucleoproteins (snRNPs) in vitro. However, the etiological link between snRNPs and SMA is unclear. We have developed a Drosophila melanogaster system to model SMA in vivo. Larval-lethal Smn-null mutations show no detectable snRNP reduction, making it unlikely that these animals die from global snRNP deprivation. Hypomorphic mutations in Smn reduce dSMN protein levels in the adult thorax, causing flightlessness and acute muscular atrophy. Mutant flight muscle motoneurons display pronounced axon routing and arborization defects. Moreover, Smn mutant myofibers fail to form thin filaments and phenocopy null mutations in Act88F, which is the flight muscle–specific actin isoform. In wild-type muscles, dSMN colocalizes with sarcomeric actin and forms a complex with α-actinin, the thin filament crosslinker. The sarcomeric localization of Smn is conserved in mouse myofibrils. These observations suggest a muscle-specific function for SMN and underline the importance of this tissue in modulating SMA severity.

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