Essential role of a trypanosome U4-specific Sm core protein in small nuclear ribonucleoprotein assembly and splicing.
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.
Jaé
Nicolas
N
Institute of Biochemistry, Justus Liebig University of Giessen, Heinrich Buff Ring 58, D-35392 Giessen, Germany.
Wang
Pingping
P
Gu
Tianpeng
T
Hühn
Martin
M
Palfi
Zsofia
Z
Urlaub
Henning
H
Bindereif
Albrecht
A
D049109:Cell Proliferation; D002499:Centrifugation, Density Gradient; D015870:Gene Expression / Q000235:genetics; D042581:Intranuclear Space / Q000378:metabolism; D013058:Mass Spectrometry; D011485:Protein Binding / Q000235:genetics; D055503:Protein Multimerization / Q000502:physiology*; D034622:RNA Interference; D012326:RNA Splicing; D012330:RNA, Double-Stranded / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*; D055517:snRNP Core Proteins / Q000235:genetics / Q000378:metabolism*
D012330:RNA, Double-Stranded; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; C405482:U4 small nuclear RNA; C405486:U6 small nuclear RNA; D055517:snRNP Core Proteins; D016914:Ribonuclease H