TbU1-70K is a U1 snRNP-specific protein and binds specifically to the 5′ loop sequence of U1 snRNA

Copyright information:

Taken from "U1 small nuclear RNP from : a minimal U1 snRNA with unusual protein components"

Nucleic Acids Research 2005;33(8):2493-2503.

Published online 29 Apr 2005

PMCID:PMC1087902.

© The Author 2005. Published by Oxford University Press. All rights reserved

() Comparison of the domain structures of (Tb08.4A8.530) and the human U1-70K (A25707) proteins. () Western blot analysis of U1 snRNP proteins. U1 snRNPs were affinity-purified from extract by a 2′--methyl RNA antisense oligonucleotide, protein was prepared and analyzed by SDS–PAGE and western blotting, using polyclonal rabbit antibodies against TbU1-70K (U1-70K) or non-immune serum (NIS). The arrow points to the immunostained TbU1-70K band of apparent molecular weight 40 kDa. Protein markers are on the right (in kDa). () U1 snRNA is specifically coprecipitated from extract by anti-Tb U1-70 antibodies. Immunoprecipitations were carried out from extract, using NIS, or with antibodies against the TbU1-70K protein (U1-70K) or against the trypanosome Sm proteins (Sm). RNA was purified from the immunoprecipitates and analyzed by 3′ end labeling with [P]pCp. The positions of the SL RNA and snRNAs are marked on the right. , P-labeled pBR322/HpaII markers. () RNA from the same immunoprecipitates was also analyzed by primer extension with a U1-specific oligonucleotide. In addition, RNA from a 10% aliquot of the input was included; the positions of the primer () and the U1-specific primer-extension product (U1) are marked on the right. , P-labeled pBR322/HpaII markers. () P-labeled U1 snRNA and mutant derivatives [as indicated above the lanes; see (F)] were transcribed and incubated with GST-TbU1-70K, followed by GST pull-down. For each reaction, 10% of the input () and the total precipitated material () were analyzed. , P-labeled pBR322/HpaII markers. () Sequences and proposed secondary structures of the U1 snRNA and its mutant derivatives. The boxed sequence in the U1 snRNA indicates the Sm site; the two arrows indicate a potential second stem–loop. Below, the sequences of the stem–loop derivatives are given; the circled nucleotides mark the two positions in the human loop that differ from the sequence, and the single-nucleotide mutation (A21) in the mutant human loop.