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

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

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

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Principle and potential outcomes of the single TaqMan-qPCR.

Principle and potential outcomes of the single TaqMan-qPCR.</p

Single TaqMan-qPCR for determining the copy number of integrated <i>mdhfr-ts</i> selectable marker.

Standard curves were made through a triplicate test of 10-fold serial dilutions of (A) P972 or (B) T. gondii RH DNA. (C) For each WT or KO clone, the number of existing dhfr-ts in the genome was determined according to the plasmid based standard curve (black bars) and the T. gondii RH DNA-based calibrator (grey bars). Since in the T. gondii genome the wtdhfr-ts is a single copy gene, the following equation was used: one WT tachyzoite = one-copy dhfr-ts, for the calculation based on T. gondii RH DNA based calibrator curve (grey bars). Error bars indicate standard deviation of triplicates for each sample. In (D), the number of inserted mdhfr-ts in each KO clone is defined by subtracting the dhfr-ts copy number found in the WT from the dhfr-ts copy number in the KO (black bars) or by subtracting the tachyzoite numbers determined for the WT from tachyzoite numbers corresponding the KO clone (grey bars). The optimal result of 1 indicates a single integration event of the mdhfr-ts into sag1.</p

Loss of <i>sag1</i> expression in <i>T</i>. <i>gondii</i> RH SAG1 knockouts by (A) Western blot analysis and (B) immunofluorescence.

Loss of sag1 expression in T. gondii RH SAG1 knockouts by (A) Western blot analysis and (B) immunofluorescence.</p

Principle and potential outcome of the duplex TaqMan-qPCR.

Principle and potential outcome of the duplex TaqMan-qPCR.</p

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SAG1 gene disruption in <i>T</i>. <i>gondii</i> RH by CRISPR-Cas9 technology.

(A) Schematic representation of the strategy used to disrupt sag1 by inserting the pyrimethamine-resistance gene MDHFR-TS. (B) Diagnostic PCR revealing integration of a complete mdhfr-ts sequence into sag1 in four clones (C18, C23, C30 and C33) compared with the parental strain RH. The KO clone C31 showed a smaller band, clones C6 and 7 exhibited a band ≤ 1000 bp. The WT locus produced the expected PCR product (~ 216 bp).</p