Limits...
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.

Show MeSH

Related in: MedlinePlus

SMN-Gemin2 cooperates with U1-70K in Sm protein recruitment(a) In vitro binding of GST-U1-70K N99 to Gemin2 and Sm proteins. Each Sm protein subunit, combinations of the subunits (lanes 1–7), Gemin2 only (lane 8) or Gemin2 with combinations of Sm protein subunit (lanes 9–15) are indicated. The input panel shows 10% of the proteins used for binding and the gel was visualized by silver staining. Uncropped images are shown in Supplementary Data Set 1. (b) Superimposed crystal structures of U1 snRNP12,14,17 and SMN-Gemin2-Sm525, showing cooperative roles in Sm core assembly intermediate of U1. Left, This Gemin2-Sm5-U1-70K intermediate contacts with pre-U1 bound by U1-70K’s SL1 RBD, places pre-U1’s Sm site at the Sm5’s inner ring and prevents binding of other RNAs. U1-70K’s N-terminal domain binds directly to BD3, which helps in their recruitment and proper positioning for Sm ring closure. For clarity, two other U1 snRNP specific proteins, U1A, which binds to SL2, and U1C, which binds to U1-70K and U1 snRNA, are not shown. Right, A rotated view compared with left, showing Gemin2 and U1-70K assist Sm protein recruitment. Gemin2 binds D1D2 through its C-terminal domain and FEG via its N-terminal domain. The loop connecting these two domains shown in a dashed line is disordered and its length (aa70–82) is likely sufficient to avoid a clash with U1-70K’s D2F binding. Two patches (aa10–31 and aa39–59) of N-terminal U1-70K bind at the BD3 and D2F interface, respectively. The Figures were prepared with PyMOL (http://www.pymol.org/).
© Copyright Policy - permissions-link
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4834709&req=5

Figure 4: SMN-Gemin2 cooperates with U1-70K in Sm protein recruitment(a) In vitro binding of GST-U1-70K N99 to Gemin2 and Sm proteins. Each Sm protein subunit, combinations of the subunits (lanes 1–7), Gemin2 only (lane 8) or Gemin2 with combinations of Sm protein subunit (lanes 9–15) are indicated. The input panel shows 10% of the proteins used for binding and the gel was visualized by silver staining. Uncropped images are shown in Supplementary Data Set 1. (b) Superimposed crystal structures of U1 snRNP12,14,17 and SMN-Gemin2-Sm525, showing cooperative roles in Sm core assembly intermediate of U1. Left, This Gemin2-Sm5-U1-70K intermediate contacts with pre-U1 bound by U1-70K’s SL1 RBD, places pre-U1’s Sm site at the Sm5’s inner ring and prevents binding of other RNAs. U1-70K’s N-terminal domain binds directly to BD3, which helps in their recruitment and proper positioning for Sm ring closure. For clarity, two other U1 snRNP specific proteins, U1A, which binds to SL2, and U1C, which binds to U1-70K and U1 snRNA, are not shown. Right, A rotated view compared with left, showing Gemin2 and U1-70K assist Sm protein recruitment. Gemin2 binds D1D2 through its C-terminal domain and FEG via its N-terminal domain. The loop connecting these two domains shown in a dashed line is disordered and its length (aa70–82) is likely sufficient to avoid a clash with U1-70K’s D2F binding. Two patches (aa10–31 and aa39–59) of N-terminal U1-70K bind at the BD3 and D2F interface, respectively. The Figures were prepared with PyMOL (http://www.pymol.org/).

Mentions: N99 directly bound BD3, but not D1D2, or FEG (Fig. 4a, lanes 1–7). In contrast, and as shown previously, Gemin2 did not bind BD3 nor did it bind D1D2 or FEG alone, however, it formed a stable complex with Sm pentamer D1D2-FEG (Sm5)24,25. SMN bound Gemin2-Sm5 is a key intermediate in Sm core assembly, and we therefore investigated if Gemin2 and U1-70K could collaborate in binding Sm. This showed that N99 did not bind Gemin2 (Fig. 4a, lane 8), however complexes containing N99, Gemin2 and Sm5 or all 7 Sm proteins (Sm7) readily formed (Fig. 4a, lanes 12 and 15). The Sm7-containing complex would not be expected to form a stable Sm ring without an RNA’s Sm site15. Thus, U1-70K and Gemin2 can cooperate to recruit all of the Sm core’s proteins, which neither alone could achieve.


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)

SMN-Gemin2 cooperates with U1-70K in Sm protein recruitment(a) In vitro binding of GST-U1-70K N99 to Gemin2 and Sm proteins. Each Sm protein subunit, combinations of the subunits (lanes 1–7), Gemin2 only (lane 8) or Gemin2 with combinations of Sm protein subunit (lanes 9–15) are indicated. The input panel shows 10% of the proteins used for binding and the gel was visualized by silver staining. Uncropped images are shown in Supplementary Data Set 1. (b) Superimposed crystal structures of U1 snRNP12,14,17 and SMN-Gemin2-Sm525, showing cooperative roles in Sm core assembly intermediate of U1. Left, This Gemin2-Sm5-U1-70K intermediate contacts with pre-U1 bound by U1-70K’s SL1 RBD, places pre-U1’s Sm site at the Sm5’s inner ring and prevents binding of other RNAs. U1-70K’s N-terminal domain binds directly to BD3, which helps in their recruitment and proper positioning for Sm ring closure. For clarity, two other U1 snRNP specific proteins, U1A, which binds to SL2, and U1C, which binds to U1-70K and U1 snRNA, are not shown. Right, A rotated view compared with left, showing Gemin2 and U1-70K assist Sm protein recruitment. Gemin2 binds D1D2 through its C-terminal domain and FEG via its N-terminal domain. The loop connecting these two domains shown in a dashed line is disordered and its length (aa70–82) is likely sufficient to avoid a clash with U1-70K’s D2F binding. Two patches (aa10–31 and aa39–59) of N-terminal U1-70K bind at the BD3 and D2F interface, respectively. The Figures were prepared with PyMOL (http://www.pymol.org/).
© Copyright Policy - permissions-link
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4834709&req=5

Figure 4: SMN-Gemin2 cooperates with U1-70K in Sm protein recruitment(a) In vitro binding of GST-U1-70K N99 to Gemin2 and Sm proteins. Each Sm protein subunit, combinations of the subunits (lanes 1–7), Gemin2 only (lane 8) or Gemin2 with combinations of Sm protein subunit (lanes 9–15) are indicated. The input panel shows 10% of the proteins used for binding and the gel was visualized by silver staining. Uncropped images are shown in Supplementary Data Set 1. (b) Superimposed crystal structures of U1 snRNP12,14,17 and SMN-Gemin2-Sm525, showing cooperative roles in Sm core assembly intermediate of U1. Left, This Gemin2-Sm5-U1-70K intermediate contacts with pre-U1 bound by U1-70K’s SL1 RBD, places pre-U1’s Sm site at the Sm5’s inner ring and prevents binding of other RNAs. U1-70K’s N-terminal domain binds directly to BD3, which helps in their recruitment and proper positioning for Sm ring closure. For clarity, two other U1 snRNP specific proteins, U1A, which binds to SL2, and U1C, which binds to U1-70K and U1 snRNA, are not shown. Right, A rotated view compared with left, showing Gemin2 and U1-70K assist Sm protein recruitment. Gemin2 binds D1D2 through its C-terminal domain and FEG via its N-terminal domain. The loop connecting these two domains shown in a dashed line is disordered and its length (aa70–82) is likely sufficient to avoid a clash with U1-70K’s D2F binding. Two patches (aa10–31 and aa39–59) of N-terminal U1-70K bind at the BD3 and D2F interface, respectively. The Figures were prepared with PyMOL (http://www.pymol.org/).
Mentions: N99 directly bound BD3, but not D1D2, or FEG (Fig. 4a, lanes 1–7). In contrast, and as shown previously, Gemin2 did not bind BD3 nor did it bind D1D2 or FEG alone, however, it formed a stable complex with Sm pentamer D1D2-FEG (Sm5)24,25. SMN bound Gemin2-Sm5 is a key intermediate in Sm core assembly, and we therefore investigated if Gemin2 and U1-70K could collaborate in binding Sm. This showed that N99 did not bind Gemin2 (Fig. 4a, lane 8), however complexes containing N99, Gemin2 and Sm5 or all 7 Sm proteins (Sm7) readily formed (Fig. 4a, lanes 12 and 15). The Sm7-containing complex would not be expected to form a stable Sm ring without an RNA’s Sm site15. Thus, U1-70K and Gemin2 can cooperate to recruit all of the Sm core’s proteins, which neither alone could achieve.

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