Reconstitution of the human U sn
            <scp>RNP</scp>
            assembly machinery reveals stepwise Sm protein organization

Neuenkirchen, Nils; Englbrecht, Clemens; Ohmer, Jürgen; Ziegenhals, Thomas; Chari, Ashwin; Fischer, Utz

doi:10.15252/embj.201490350

Cited by 50 publications

(67 citation statements)

References 63 publications

(137 reference statements)

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“…However, it is not impossible that an as yet unidentified RNA-binding protein unrelated to Gemin5 may transiently associate with the SMN complex to fulfill the role of Gemin5. Another concern is that Gemin5 appears to be not needed for snRNP assembly in vitro (Neuenkirchen et al 2015). There are several possible explanations for this observation.…”

Section: Discussionmentioning

confidence: 99%

“…A recent study revealed a special U1 snRNP assembly pathway dependent on the U1-70K protein, which binds SL1 (So et al 2016). This finding perhaps explains the observation that Gemin5 is not strictly needed for snRNP assembly in vitro, judged by an assay using purified systems (Neuenkirchen et al 2015). The binding of the SMN complex and Gemin5 to the Sm site is sequence-specific, but the nucleotide sequence for the stem-loop appears to be unimportant.…”

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confidence: 89%

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Structural basis for snRNA recognition by the double-WD40 repeat domain of Gemin5

et al. 2016

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Assembly of the spliceosomal small nuclear ribonucleoparticle (snRNP) core requires the participation of the multisubunit SMN (survival of motor neuron) complex, which contains SMN and several Gemin proteins. The SMN and Gemin2 subunits directly bind Sm proteins, and Gemin5 is required for snRNP biogenesis and has been implicated in snRNA recognition. The RNA sequence required for snRNP assembly includes the Sm site and an adjacent 3 ′ stem-loop, but a precise understanding of Gemin5's RNA-binding specificity is lacking. Here we show that the N-terminal half of Gemin5, which is composed of two juxtaposed seven-bladed WD40 repeat domains, recognizes the Sm site. The tandem WD40 repeat domains are rigidly held together to form a contiguous RNA-binding surface. RNA-contacting residues are located mostly on loops between β strands on the apical surface of the WD40 domains. Structural and biochemical analyses show that base-stacking interactions involving four aromatic residues and hydrogen bonding by a pair of arginines are crucial for specific recognition of the Sm sequence. We also show that an adenine immediately 5 ′ to the Sm site is required for efficient binding and that Gemin5 can bind short RNA oligos in an alternative mode. Our results provide mechanistic understandings of Gemin5's snRNA-binding specificity as well as valuable insights into the molecular mechanism of RNA binding by WD40 repeat proteins in general.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 89%

Structural basis for snRNA recognition by the double-WD40 repeat domain of Gemin5

et al. 2016

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“…Whereas other members of the pathway monomethylate or asymmetrically dimethylate arginine residues, PRMT5 symmetrically dimethylates arginine residues (reviewed by Bedford and Clarke, 2009;Stopa et al, 2015). Regulating a diverse set of target proteins, it can methylate specific Sm proteins, facilitating their assembly into a core complex of spliceosomal snRNPs (Chari et al, 2008;Meister and Fischer, 2002;Neuenkirchen et al, 2015;Pelz et al, 2015). Additionally, PRMT5 can regulate gene expression by methylation of histones H2A/H4 and H3 (specifically H2A/H4R3 and H3R8), which are generally associated with transcriptional repression (Ancelin et al, 2006;Di Lorenzo and Bedford, 2011;Lee and Bedford, 2002;Tee et al, 2010;Zhang et al, 2015;Zhao et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud

Norrie

et al. 2016

Development

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During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4. Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis.

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“…The assembly of the human Sm ring around the Sm RNA site is an intricate and ordered process mediated by the SMN complex, comprising the SMN (survival motor neuron) protein and several Gemin proteins, and other factors (Liu et al 1997;Zhang et al 2011;Grimm et al 2013;Neuenkirchen et al 2015). Mutations in the human SMN1 gene are the cause of the disease spinal muscular atrophy (Lunn and Wang 2008;Chari et al 2009).…”

Section: Introductionmentioning

confidence: 99%

Structure–function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring

Schwer

Kruchten

Shuman

2016

RNA

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A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure-function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein-RNA and protein-protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not.

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Reconstitution of the human U sn RNP assembly machinery reveals stepwise Sm protein organization

Cited by 50 publications

References 63 publications

Structural basis for snRNA recognition by the double-WD40 repeat domain of Gemin5

Structural basis for snRNA recognition by the double-WD40 repeat domain of Gemin5

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud

Structure–function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring

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