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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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The U1-snRNP-specific stem-loop 1 binding protein U1-70K bridges pre-U1 or U1 snRNA to the SMN complex independent of Gemin5(a) Western blot analysis of SMN complex components bound to biotinylated U4 and U4ΔSm snRNAs in HeLa cells from control, U1-70K, SMN or Gemin5 siRNA knockdowns. The input lanes show 20% of each of the cell extracts used. The knockdowns’ efficiencies relative to Magoh as loading control are indicated as % of residual protein for each knockdown compared to control (100%). A schematic illustration of U4 structure and its canonical Sm site in snRNP code, which is the RNA sequence necessary and sufficient for Gemin5 binding, are indicated. (b) The same western blot analysis as in a for biotinylated U1, including U1 precursor (pre-U1), U1ΔSm, SL1 mutant (U1A3) and super-U1. A U30C) in the SL1, which abolishes U1-70K binding. Super-U1 is a mutation that replaces U1’s Sm site with a canonical Sm site. Uncropped scans of western blots are shown in Supplementary Data Set 1.
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Figure 1: The U1-snRNP-specific stem-loop 1 binding protein U1-70K bridges pre-U1 or U1 snRNA to the SMN complex independent of Gemin5(a) Western blot analysis of SMN complex components bound to biotinylated U4 and U4ΔSm snRNAs in HeLa cells from control, U1-70K, SMN or Gemin5 siRNA knockdowns. The input lanes show 20% of each of the cell extracts used. The knockdowns’ efficiencies relative to Magoh as loading control are indicated as % of residual protein for each knockdown compared to control (100%). A schematic illustration of U4 structure and its canonical Sm site in snRNP code, which is the RNA sequence necessary and sufficient for Gemin5 binding, are indicated. (b) The same western blot analysis as in a for biotinylated U1, including U1 precursor (pre-U1), U1ΔSm, SL1 mutant (U1A3) and super-U1. A U30C) in the SL1, which abolishes U1-70K binding. Super-U1 is a mutation that replaces U1’s Sm site with a canonical Sm site. Uncropped scans of western blots are shown in Supplementary Data Set 1.

Mentions: To understand the role of U1’s SL1 and snRNP code in Sm core assembly, we compared the binding of SMN complex components (SMN, Gemin2–8 and Unrip) to biotinylated pre-U1, mature U1, U1A3 (a U1 SL1 mutant defective in the U1-snRNP specific U1-70K binding)28, U1ΔSm (a U1 Sm site mutant that cannot assemble Sm core), super-U1 (a chimeric U1 in which the Sm site was replaced by a canonical Sm site, AUUUUUG), U4 snRNA (U4), a canonical snRNP code containing snRNA, and U4ΔSm. The RNAs were incubated in human (HeLa) cell extracts in which either SMN, Gemin5 or U1-70K had been knocked down by RNAi (Fig. 1a, input). Although U1-70K has not been previously suggested to have a role in Sm core assembly, we tested its effect because it is the only protein known to bind SL128,29. For most experiments we used pre-U1, which is 50nt longer at the 3′-end than U1, because it represents the U1 substrate for Sm core assembly in cells20. For U4, whose precursor is only 6nt longer than mature U4, there is little difference between the precursor and mature forms (data not shown).


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)

The U1-snRNP-specific stem-loop 1 binding protein U1-70K bridges pre-U1 or U1 snRNA to the SMN complex independent of Gemin5(a) Western blot analysis of SMN complex components bound to biotinylated U4 and U4ΔSm snRNAs in HeLa cells from control, U1-70K, SMN or Gemin5 siRNA knockdowns. The input lanes show 20% of each of the cell extracts used. The knockdowns’ efficiencies relative to Magoh as loading control are indicated as % of residual protein for each knockdown compared to control (100%). A schematic illustration of U4 structure and its canonical Sm site in snRNP code, which is the RNA sequence necessary and sufficient for Gemin5 binding, are indicated. (b) The same western blot analysis as in a for biotinylated U1, including U1 precursor (pre-U1), U1ΔSm, SL1 mutant (U1A3) and super-U1. A U30C) in the SL1, which abolishes U1-70K binding. Super-U1 is a mutation that replaces U1’s Sm site with a canonical Sm site. Uncropped scans of western blots are shown in Supplementary Data Set 1.
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Related In: Results  -  Collection

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Figure 1: The U1-snRNP-specific stem-loop 1 binding protein U1-70K bridges pre-U1 or U1 snRNA to the SMN complex independent of Gemin5(a) Western blot analysis of SMN complex components bound to biotinylated U4 and U4ΔSm snRNAs in HeLa cells from control, U1-70K, SMN or Gemin5 siRNA knockdowns. The input lanes show 20% of each of the cell extracts used. The knockdowns’ efficiencies relative to Magoh as loading control are indicated as % of residual protein for each knockdown compared to control (100%). A schematic illustration of U4 structure and its canonical Sm site in snRNP code, which is the RNA sequence necessary and sufficient for Gemin5 binding, are indicated. (b) The same western blot analysis as in a for biotinylated U1, including U1 precursor (pre-U1), U1ΔSm, SL1 mutant (U1A3) and super-U1. A U30C) in the SL1, which abolishes U1-70K binding. Super-U1 is a mutation that replaces U1’s Sm site with a canonical Sm site. Uncropped scans of western blots are shown in Supplementary Data Set 1.
Mentions: To understand the role of U1’s SL1 and snRNP code in Sm core assembly, we compared the binding of SMN complex components (SMN, Gemin2–8 and Unrip) to biotinylated pre-U1, mature U1, U1A3 (a U1 SL1 mutant defective in the U1-snRNP specific U1-70K binding)28, U1ΔSm (a U1 Sm site mutant that cannot assemble Sm core), super-U1 (a chimeric U1 in which the Sm site was replaced by a canonical Sm site, AUUUUUG), U4 snRNA (U4), a canonical snRNP code containing snRNA, and U4ΔSm. The RNAs were incubated in human (HeLa) cell extracts in which either SMN, Gemin5 or U1-70K had been knocked down by RNAi (Fig. 1a, input). Although U1-70K has not been previously suggested to have a role in Sm core assembly, we tested its effect because it is the only protein known to bind SL128,29. For most experiments we used pre-U1, which is 50nt longer at the 3′-end than U1, because it represents the U1 substrate for Sm core assembly in cells20. For U4, whose precursor is only 6nt longer than mature U4, there is little difference between the precursor and mature forms (data not shown).

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