Essential Role of a Trypanosome U4-Specific Sm Core Protein in Small Nuclear Ribonucleoprotein Assembly and Splicing

Abstract

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.The excision of intronic sequences from precursor mRNAs is a critical step during eukaryotic gene expression. This reaction is catalyzed by the spliceosome, a macromolecular complex composed of small nuclear ribonucleoproteins (snRNPs) and many additional proteins. Spliceosome assembly and splicing catalysis occur in an ordered multistep process, which includes multiple conformational rearrangements (35). Spliceosomal snRNPs are assembled from snRNAs and protein components, the latter of which fall into two classes: snRNPspecific and common proteins. The common or canonical core proteins are also termed Sm proteins, specifically SmB, SmD1, SmD2, SmD3, SmE, SmF, and SmG (10; reviewed in reference 9), which all share an evolutionarily conserved bipartite sequence motif (Sm1 and Sm2) required for Sm protein interactions and the formation of the heteroheptameric Sm core complex around the Sm sites of the snRNAs (3,7,29). Prior to this, the Sm proteins form three heteromeric subcomplexes: SmD3/SmB, SmD1/SmD2, and SmE/SmF/SmG (23; reviewed in reference 34). Individual Sm proteins or Sm subcomplexes cannot stably interact with the snRNA. Instead, a stable subcore forms by an association of the subcomplexes SmD1/SmD2 and SmE/SmF/SmG with the Sm site on the snRNA; the subsequent integration of the SmD3/SmB heterodimer completes Sm core assembly.In addition to the canonical Sm proteins, other proteins carrying the Sm motif have been identified for many eukaryotes. Those proteins, termed LSm (like Sm) proteins, exist in distinct heptameric complexes that differ in function and localization. For example, a complex composed of LSm1 to LSm7 (LSm1-7) accumulates in cytoplasmic foci and participates in mRNA turnover (4,8,31). Another complex, LSm2-8, binds to the 3Ј oligo(U) tract of the U6 snRNA in the nucleus (1,15,24). Finally, in the U7 snRNP, which is involved in histone mRNA 3Ј-end processing, the Sm proteins SmD1 and SmD2 are replaced by U7-specific LSm10 and LSm11 proteins, respectively (20, 21; reviewed in reference 28).This knowledge is based primarily on the mammalian system, where spliceosomal snRN...

snRNA-specific role of SMN in trypanosome snRNP biogenesis in vivo

“…18 A similar case was subsequently found in the trypanosome U4 snRNP. 16,19 These variations impose an additional complication on the specificity of Sm core assembly in trypanosomes. Searching for potential specificity factors involved, we could identify a very divergent SMN-homologous protein in T. brucei, which strongly deviates in its domain organization from higher eukaryotes; in particular, it lacks the central Tudor domain.…”

“…Regarding U4 and U6, we observed a dramatic change after SMN knockdown: The CsCl-stable U4/U6 Sm core complex conditions. 19,[39][40][41] Although SL RNA total levels increased severalfold after SMN knockdown (compare input lanes), only a minor portion of SL RNA remained in the form of stable core complexes (fractions #2-3) and most of the SL RNA sedimented at From uninduced cells (t0) and after 1, 2 and 3 days of RNAi silencing (t1-t3) lysates were prepared and subjected to anti-sm immunoprecipitation. To assess immunoprecipitation efficiencies, RNA from 15% input (lanes 1-4) and total anti-sm immunoprecipitates (lanes 5-8) were analyzed by northern blotting for U2, sL, U4, U6, U1 and U5 snRNAs (the latter two from a longer exposure of the same blot; (A); see (B) for quantifications; U1 and U5 snRNAs could not be quantitated because very long exposure times were necessary).…”

The Pre-mRNA Splicing Machinery of Trypanosomes: Complex or Simplified?

“…Alternatively, the specific interactions of the U2 Sm paralogs SmK15/ SmK16.5 with U2AЈ/U2BЉ may facilitate correct assembly of the U2-specific Sm complex onto the U2 Sm binding site. On the other hand, U4 core snRNP formation appears to be independent of snRNP-specific proteins, because in core RNP reconstitution assays, SSm4 alone determined efficient and specific assembly of the U4-specific Sm complex onto the U4 snRNA (30).…”

“…Importantly, the study by Tkacz et al (83) provided an in vivo analysis demonstrating that RNAi-mediated expression silencing of the specific Sm paralogs reduced the abundance of only the corresponding U snRNA. The U4-specific Sm core was subsequently characterized at the biochemical level, verifying the U4 association of the SmD3 paralog (30). Unfortunately, the studies on Sm core variation established different nomenclatures, and Sm15K is also referred to as specific spliceosomal Sm2-1 protein (SSm2-1), Sm16.5K as SSm2-2, and the U4-specific protein as SSm4.…”

“…Tb927.3.3160), encoding poly(A) polymerase, was shown to harbor a single intron that was removed by conventional cis splicing (48). The search for further introns revealed only one more gene in T. brucei (Tb927.8.1510), encoding a putative RNA helicase (7), whose pre-mRNA was shown to be cis spliced (30). Interestingly, a recent characterization of the T. brucei transcriptome by high-throughput RNA sequencing strongly indicates that there are no other introns disrupting protein-coding genes (75).…”