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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 bridges pre-U1 snRNA to SMN and mediates preferential Sm core assembly on U1 snRNA over other snRNAs(a) Western blot of SMN complex proteins bound to the biotinylated pre-U1 snRNA in U1-70K knockdown cell extracts complemented with recombinant U1-70K N194. The input lane shows 20% of the cell extracts and the N194 used for binding. (b) In vitro Sm core assembly activities on pre-U1 and U4 with increasing concentrations of N194 in U1-70K knockdown extracts. The rescued snRNP assembly capacities on snRNAs were determined by % changes from the U1-70K knockdown without N194. The error bars represent standard deviation from three independent biological replicates. (c) Binding of GST-U1-70K proteins to recombinant SMN protein. Schematic diagram of U1-70K and its deletion fragments with corresponding residue numbers are indicated. The input lane shows 10% of the SMN protein used for binding and the gel was visualized by Simplyblue staining. (d) Binding of GST-N194 to SMN in the absences or presence of equimolar amount of in vitro transcribed pre-U1. The protein gel was visualized by silver staining. (e) Binding of GST-N90–N194 protein to in vitro-translated [35S]-methionine labeled wild-type (WT) SMN and its domain deletions. The input lanes show 10% of SMN’s deletion domains (N91, aa1–91; Tudor, aa92–158; Exon7, aa279–294; C106, aa189–294) and the gel was visualized by autoradiography. (f) snRNP assembly measurements as in b with N194 deletion fragments using 1.25 molar ratio of U1-70K proteins to snRNA. The error bars represent standard deviation from three independent biological replicates. Uncropped scans of blots, gels and autoradiographs are shown in Supplementary Data Set 1.
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Figure 3: U1-70K bridges pre-U1 snRNA to SMN and mediates preferential Sm core assembly on U1 snRNA over other snRNAs(a) Western blot of SMN complex proteins bound to the biotinylated pre-U1 snRNA in U1-70K knockdown cell extracts complemented with recombinant U1-70K N194. The input lane shows 20% of the cell extracts and the N194 used for binding. (b) In vitro Sm core assembly activities on pre-U1 and U4 with increasing concentrations of N194 in U1-70K knockdown extracts. The rescued snRNP assembly capacities on snRNAs were determined by % changes from the U1-70K knockdown without N194. The error bars represent standard deviation from three independent biological replicates. (c) Binding of GST-U1-70K proteins to recombinant SMN protein. Schematic diagram of U1-70K and its deletion fragments with corresponding residue numbers are indicated. The input lane shows 10% of the SMN protein used for binding and the gel was visualized by Simplyblue staining. (d) Binding of GST-N194 to SMN in the absences or presence of equimolar amount of in vitro transcribed pre-U1. The protein gel was visualized by silver staining. (e) Binding of GST-N90–N194 protein to in vitro-translated [35S]-methionine labeled wild-type (WT) SMN and its domain deletions. The input lanes show 10% of SMN’s deletion domains (N91, aa1–91; Tudor, aa92–158; Exon7, aa279–294; C106, aa189–294) and the gel was visualized by autoradiography. (f) snRNP assembly measurements as in b with N194 deletion fragments using 1.25 molar ratio of U1-70K proteins to snRNA. The error bars represent standard deviation from three independent biological replicates. Uncropped scans of blots, gels and autoradiographs are shown in Supplementary Data Set 1.

Mentions: Delineation of U1-70K’s domains showed that U1-70K’s C-terminal arginine- and serine-rich (RS) domain (aa230–437), which is important for splicing34, was not required for bridging pre-U1 to the SMN complex (data not shown). However, recombinant U1-70K’s N-terminal 194 amino acids (N194) restored the SMN complex’s association with pre-U1 in U1-70K knockdown cell extracts (Fig. 3a). Furthermore, N194 bound SMN directly and enhanced pre-U1- and inhibited U4-Sm core assembly (Fig. 3b, c). The smallest N194 fragment that was necessary and sufficient to bind SMN was N90-194, which contains U1-70K’s SL1-specific RNA-binding domain (RBD/RRM; aa102–181)29, while the N-terminal domain lacking the RBD, N99 (aa1–99) did not bind SMN (Fig. 3c). Moreover, pre-U1 enhanced N194 binding to SMN (2.1 fold) (Fig. 3d). Thus, pre-U1 binding creates additional interactions between N194 and SMN, either by an RNA-mediated allosteric effect that increases U1-70K interaction surface with SMN or by additional binding interactions between pre-U1 with SMN. The ability of the same type of RBD as U1-70K’s to engage in protein interactions through a surface other than the RNA binding site has been described for other RBPs35,36.


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 bridges pre-U1 snRNA to SMN and mediates preferential Sm core assembly on U1 snRNA over other snRNAs(a) Western blot of SMN complex proteins bound to the biotinylated pre-U1 snRNA in U1-70K knockdown cell extracts complemented with recombinant U1-70K N194. The input lane shows 20% of the cell extracts and the N194 used for binding. (b) In vitro Sm core assembly activities on pre-U1 and U4 with increasing concentrations of N194 in U1-70K knockdown extracts. The rescued snRNP assembly capacities on snRNAs were determined by % changes from the U1-70K knockdown without N194. The error bars represent standard deviation from three independent biological replicates. (c) Binding of GST-U1-70K proteins to recombinant SMN protein. Schematic diagram of U1-70K and its deletion fragments with corresponding residue numbers are indicated. The input lane shows 10% of the SMN protein used for binding and the gel was visualized by Simplyblue staining. (d) Binding of GST-N194 to SMN in the absences or presence of equimolar amount of in vitro transcribed pre-U1. The protein gel was visualized by silver staining. (e) Binding of GST-N90–N194 protein to in vitro-translated [35S]-methionine labeled wild-type (WT) SMN and its domain deletions. The input lanes show 10% of SMN’s deletion domains (N91, aa1–91; Tudor, aa92–158; Exon7, aa279–294; C106, aa189–294) and the gel was visualized by autoradiography. (f) snRNP assembly measurements as in b with N194 deletion fragments using 1.25 molar ratio of U1-70K proteins to snRNA. The error bars represent standard deviation from three independent biological replicates. Uncropped scans of blots, gels and autoradiographs are shown in Supplementary Data Set 1.
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Figure 3: U1-70K bridges pre-U1 snRNA to SMN and mediates preferential Sm core assembly on U1 snRNA over other snRNAs(a) Western blot of SMN complex proteins bound to the biotinylated pre-U1 snRNA in U1-70K knockdown cell extracts complemented with recombinant U1-70K N194. The input lane shows 20% of the cell extracts and the N194 used for binding. (b) In vitro Sm core assembly activities on pre-U1 and U4 with increasing concentrations of N194 in U1-70K knockdown extracts. The rescued snRNP assembly capacities on snRNAs were determined by % changes from the U1-70K knockdown without N194. The error bars represent standard deviation from three independent biological replicates. (c) Binding of GST-U1-70K proteins to recombinant SMN protein. Schematic diagram of U1-70K and its deletion fragments with corresponding residue numbers are indicated. The input lane shows 10% of the SMN protein used for binding and the gel was visualized by Simplyblue staining. (d) Binding of GST-N194 to SMN in the absences or presence of equimolar amount of in vitro transcribed pre-U1. The protein gel was visualized by silver staining. (e) Binding of GST-N90–N194 protein to in vitro-translated [35S]-methionine labeled wild-type (WT) SMN and its domain deletions. The input lanes show 10% of SMN’s deletion domains (N91, aa1–91; Tudor, aa92–158; Exon7, aa279–294; C106, aa189–294) and the gel was visualized by autoradiography. (f) snRNP assembly measurements as in b with N194 deletion fragments using 1.25 molar ratio of U1-70K proteins to snRNA. The error bars represent standard deviation from three independent biological replicates. Uncropped scans of blots, gels and autoradiographs are shown in Supplementary Data Set 1.
Mentions: Delineation of U1-70K’s domains showed that U1-70K’s C-terminal arginine- and serine-rich (RS) domain (aa230–437), which is important for splicing34, was not required for bridging pre-U1 to the SMN complex (data not shown). However, recombinant U1-70K’s N-terminal 194 amino acids (N194) restored the SMN complex’s association with pre-U1 in U1-70K knockdown cell extracts (Fig. 3a). Furthermore, N194 bound SMN directly and enhanced pre-U1- and inhibited U4-Sm core assembly (Fig. 3b, c). The smallest N194 fragment that was necessary and sufficient to bind SMN was N90-194, which contains U1-70K’s SL1-specific RNA-binding domain (RBD/RRM; aa102–181)29, while the N-terminal domain lacking the RBD, N99 (aa1–99) did not bind SMN (Fig. 3c). Moreover, pre-U1 enhanced N194 binding to SMN (2.1 fold) (Fig. 3d). Thus, pre-U1 binding creates additional interactions between N194 and SMN, either by an RNA-mediated allosteric effect that increases U1-70K interaction surface with SMN or by additional binding interactions between pre-U1 with SMN. The ability of the same type of RBD as U1-70K’s to engage in protein interactions through a surface other than the RNA binding site has been described for other RBPs35,36.

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