U1C (TbU1C): a U1 snRNP-specific component binding specifically to the 5′ terminal sequence of U1 snRNA

<p><b>Copyright information:</b></p><p>Taken from "U1 small nuclear RNP from : a minimal U1 snRNA with unusual protein components"</p><p>Nucleic Acids Research 2005;33(8):2493-2503.</p><p>Published online 29 Apr 2005</p><p>PMCID:PMC1087902.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () ClustalW alignment of the protein sequences for the newly identified U1C homologs from , and , in comparison with the human U1C sequence. The conserved CH-type Zn finger within the boxed sequence is highlighted by large-size letters; asterisks indicate absolutely conserved amino acid positions. Accession numbers (GeneDB): (Tb10.70.5640), (Tc00.1047053511367.354) and (LmjF21.0320); human U1C (P09234). () Extract was prepared from a cell line, which stably expresses TAP-tagged TbU1C protein, and used to affinity-purify TAP-tagged complexes. Purification was followed by analyzing copurifying RNAs by northern blotting, using a mixed snRNA probe (snRNA positions indicated on the right). , DIG marker V (Roche). Lane 1, 1% of input; lane 2, 10% of IgG-selected and TEV-released material. Affinity-purified complexes were then immunoprecipitated with NIS (lane 3), anti TbU1-70K (lane 4) or anti-Sm antibodies (lane 5), using 30% for each immunoprecipitation. () TbU1C protein binds specifically to the 5′ terminal sequence of U1 snRNA. GST TbU1C protein was incubated with P-labeled full-length U1 snRNA (lanes 1 and 2) and various U1 snRNA derivatives: U1 Δstem–loop (lanes 3 and 4), U1 Δ5′(1–14) (lanes 5, 6), U1 Δ5′(1–30) (lanes 7 and 8), U1 5′ stem–loop (lanes 9 and 10), U1 5′(1–14) (lanes 11 and 12) or a 17mer control RNA (lanes 13 and 14). In each case, 10% of the input () and the total GST pull-down material () were analyzed

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

<p><b>Copyright information:</b></p><p>Taken from "U1 small nuclear RNP from : a minimal U1 snRNA with unusual protein components"</p><p>Nucleic Acids Research 2005;33(8):2493-2503.</p><p>Published online 29 Apr 2005</p><p>PMCID:PMC1087902.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () 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

Protein–protein interactions in the trypanosome U1 snRNP: TbU1-70K interacts with both TbU1C and TbU1-24K

<p><b>Copyright information:</b></p><p>Taken from "U1 small nuclear RNP from : a minimal U1 snRNA with unusual protein components"</p><p>Nucleic Acids Research 2005;33(8):2493-2503.</p><p>Published online 29 Apr 2005</p><p>PMCID:PMC1087902.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () GST-fusion proteins of TbU1C, TbU1-24K and TbU1-70K, as well as GST alone as a control were immobilized on glutathione-Sepharose. Corresponding aliquots of immobilized proteins were analyzed for their protein content by SDS–PAGE and Coomassie staining. The arrows point to the proteins listed above the lanes. , protein marker (in kDa). () Immobilized GST proteins (as indicated above the lanes) were incubated with S-labeled TbU1C (lanes 1–4), TbU1-24K (lanes 5–8) or TbU1-70K (lanes 9–12). After washing, bound proteins were recovered and analyzed by SDS–PAGE and fluorography. The arrows point to the respective S-labeled proteins