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A U1 snRNP-specific assembly pathway reveals the SMN complex as a versatile hub for RNP exchange.

So BR, Wan L, Zhang Z, Li P, Babiash E, Duan J, Younis I, Dreyfuss G - Nat. Struct. Mol. Biol. (2016)

Bottom Line: In Sm-core assembly, a key snRNP-biogenesis step mediated by the SMN complex, the snRNA-specific RNA-binding protein (RBP) Gemin5 delivers pre-snRNAs, which join SMN-Gemin2-recruited Sm proteins.U1-70K hijacks SMN-Gemin2-Sm, enhancing Sm-core assembly on U1s and inhibiting that on other snRNAs, thereby promoting U1 overabundance and regulating snRNP repertoire.We propose that SMN-Gemin2 is a versatile hub for RNP exchange that functions broadly in RNA metabolism.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

ABSTRACT
Despite equal snRNP stoichiometry in spliceosomes, U1 snRNP (U1) is typically the most abundant vertebrate snRNP. Mechanisms regulating U1 overabundance and snRNP repertoire are unknown. In Sm-core assembly, a key snRNP-biogenesis step mediated by the SMN complex, the snRNA-specific RNA-binding protein (RBP) Gemin5 delivers pre-snRNAs, which join SMN-Gemin2-recruited Sm proteins. We show that the human U1-specific RBP U1-70K can bridge pre-U1 to SMN-Gemin2-Sm, in a Gemin5-independent manner, thus establishing an additional and U1-exclusive Sm core-assembly pathway. U1-70K hijacks SMN-Gemin2-Sm, enhancing Sm-core assembly on U1s and inhibiting that on other snRNAs, thereby promoting U1 overabundance and regulating snRNP repertoire. SMN-Gemin2's ability to facilitate transactions between different RBPs and RNAs explains its multi-RBP valency and the myriad transcriptome perturbations associated with SMN deficiency in neurodegenerative spinal muscular atrophy. We propose that SMN-Gemin2 is a versatile hub for RNP exchange that functions broadly in RNA metabolism.

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U1-70K enhances U1 Sm core assembly, inhibits other snRNAs’ Sm core assembly in vitro, and regulates snRNP repertoire in cells(a) Quantitative in vitro Sm core assembly activities on the indicated snRNAs in extracts from cells with control, U1-70K, SMN, Gemin2 or Gemin5 siRNA knockdowns. The Sm core assembly activities on each snRNA were compared to control RNAi extracts (100% activity), except U1ΔSm and U4ΔSm, whose relative activities were compared to pre-U1 and U4, respectively. The error bars represent standard deviation from three independent biological replicates. (b) Quantitative measurements of snRNAs from the mature and nascent snRNPs in cells with control, U1-70K, SMN or Gemin5 knockdown by real-time RT-PCR. The relative amount of snRNAs compared to control RNAi are shown. The error bars represent standard deviation from three independent biological replicates.
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Figure 2: U1-70K enhances U1 Sm core assembly, inhibits other snRNAs’ Sm core assembly in vitro, and regulates snRNP repertoire in cells(a) Quantitative in vitro Sm core assembly activities on the indicated snRNAs in extracts from cells with control, U1-70K, SMN, Gemin2 or Gemin5 siRNA knockdowns. The Sm core assembly activities on each snRNA were compared to control RNAi extracts (100% activity), except U1ΔSm and U4ΔSm, whose relative activities were compared to pre-U1 and U4, respectively. The error bars represent standard deviation from three independent biological replicates. (b) Quantitative measurements of snRNAs from the mature and nascent snRNPs in cells with control, U1-70K, SMN or Gemin5 knockdown by real-time RT-PCR. The relative amount of snRNAs compared to control RNAi are shown. The error bars represent standard deviation from three independent biological replicates.

Mentions: Next, we used a quantitative snRNP assembly assay31,32 to determine the role of U1-70K and Gemin5 in Sm core assembly. (Fig. 2a and Supplementary Data Fig. 1). As expected, Sm core assembly on all tested RNAs depended on SMN-Gemin2 and required an Sm site. Gemin5 knockdown decreased Sm core assembly of canonical snRNP code-containing snRNAs (U2, U4 and U5) and to a lesser extent on SL1-containing snRNAs. Strikingly, U1-70K knockdown decreased pre-U1 assembly (by 50%), but enhanced U2, U4 and U5 Sm core assembly (up to 100%). Loss of U1-70K binding or enhancement of Gemin5 binding in U1A3 and super-U1, respectively, made them more similar to non-U1 snRNAs. These observations indicate a new role for U1-70K as a specific pre-U1 Sm core assembly factor and an inhibitor of Sm core assembly on other snRNAs.


A U1 snRNP-specific assembly pathway reveals the SMN complex as a versatile hub for RNP exchange.

So BR, Wan L, Zhang Z, Li P, Babiash E, Duan J, Younis I, Dreyfuss G - Nat. Struct. Mol. Biol. (2016)

U1-70K enhances U1 Sm core assembly, inhibits other snRNAs’ Sm core assembly in vitro, and regulates snRNP repertoire in cells(a) Quantitative in vitro Sm core assembly activities on the indicated snRNAs in extracts from cells with control, U1-70K, SMN, Gemin2 or Gemin5 siRNA knockdowns. The Sm core assembly activities on each snRNA were compared to control RNAi extracts (100% activity), except U1ΔSm and U4ΔSm, whose relative activities were compared to pre-U1 and U4, respectively. The error bars represent standard deviation from three independent biological replicates. (b) Quantitative measurements of snRNAs from the mature and nascent snRNPs in cells with control, U1-70K, SMN or Gemin5 knockdown by real-time RT-PCR. The relative amount of snRNAs compared to control RNAi are shown. The error bars represent standard deviation from three independent biological replicates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4834709&req=5

Figure 2: U1-70K enhances U1 Sm core assembly, inhibits other snRNAs’ Sm core assembly in vitro, and regulates snRNP repertoire in cells(a) Quantitative in vitro Sm core assembly activities on the indicated snRNAs in extracts from cells with control, U1-70K, SMN, Gemin2 or Gemin5 siRNA knockdowns. The Sm core assembly activities on each snRNA were compared to control RNAi extracts (100% activity), except U1ΔSm and U4ΔSm, whose relative activities were compared to pre-U1 and U4, respectively. The error bars represent standard deviation from three independent biological replicates. (b) Quantitative measurements of snRNAs from the mature and nascent snRNPs in cells with control, U1-70K, SMN or Gemin5 knockdown by real-time RT-PCR. The relative amount of snRNAs compared to control RNAi are shown. The error bars represent standard deviation from three independent biological replicates.
Mentions: Next, we used a quantitative snRNP assembly assay31,32 to determine the role of U1-70K and Gemin5 in Sm core assembly. (Fig. 2a and Supplementary Data Fig. 1). As expected, Sm core assembly on all tested RNAs depended on SMN-Gemin2 and required an Sm site. Gemin5 knockdown decreased Sm core assembly of canonical snRNP code-containing snRNAs (U2, U4 and U5) and to a lesser extent on SL1-containing snRNAs. Strikingly, U1-70K knockdown decreased pre-U1 assembly (by 50%), but enhanced U2, U4 and U5 Sm core assembly (up to 100%). Loss of U1-70K binding or enhancement of Gemin5 binding in U1A3 and super-U1, respectively, made them more similar to non-U1 snRNAs. These observations indicate a new role for U1-70K as a specific pre-U1 Sm core assembly factor and an inhibitor of Sm core assembly on other snRNAs.

Bottom Line: In Sm-core assembly, a key snRNP-biogenesis step mediated by the SMN complex, the snRNA-specific RNA-binding protein (RBP) Gemin5 delivers pre-snRNAs, which join SMN-Gemin2-recruited Sm proteins.U1-70K hijacks SMN-Gemin2-Sm, enhancing Sm-core assembly on U1s and inhibiting that on other snRNAs, thereby promoting U1 overabundance and regulating snRNP repertoire.We propose that SMN-Gemin2 is a versatile hub for RNP exchange that functions broadly in RNA metabolism.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

ABSTRACT
Despite equal snRNP stoichiometry in spliceosomes, U1 snRNP (U1) is typically the most abundant vertebrate snRNP. Mechanisms regulating U1 overabundance and snRNP repertoire are unknown. In Sm-core assembly, a key snRNP-biogenesis step mediated by the SMN complex, the snRNA-specific RNA-binding protein (RBP) Gemin5 delivers pre-snRNAs, which join SMN-Gemin2-recruited Sm proteins. We show that the human U1-specific RBP U1-70K can bridge pre-U1 to SMN-Gemin2-Sm, in a Gemin5-independent manner, thus establishing an additional and U1-exclusive Sm core-assembly pathway. U1-70K hijacks SMN-Gemin2-Sm, enhancing Sm-core assembly on U1s and inhibiting that on other snRNAs, thereby promoting U1 overabundance and regulating snRNP repertoire. SMN-Gemin2's ability to facilitate transactions between different RBPs and RNAs explains its multi-RBP valency and the myriad transcriptome perturbations associated with SMN deficiency in neurodegenerative spinal muscular atrophy. We propose that SMN-Gemin2 is a versatile hub for RNP exchange that functions broadly in RNA metabolism.

Show MeSH
Related in: MedlinePlus