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Trypanosoma cruzi proteins identified in pull-down assays aimed to characterize RNA proteins interacting with LYT1 mRNAs

ABSTRACT: This project was aimed to identify proteins interacting with the untranslated regions (UTRs) of the mRNAs transcribed from the Trypanosoma cruzi LYT1 gene. LYT1 protein is a T. cruzi virulence factor, which is expressed as two isoforms: kLYT1 and mLYT1. The proteomic data associated with this project derive from three biological replicas of pull-down assays in which particular regions of the LYT1 mRNAs (5 UTR kLYT1, 5 UTR mLYT1, and 3 UTRs types I and II) were used as baits. These RNA baits were incubated with crude extracts from either epimastigotes or trypomastigotes of the T. cruzi 058PUJ (DTU I) strain. After washing out of non-bound proteins, the remaining proteins were analyzed by liquid chromatography coupled to mass spectrometry (LC/MS). This project contains the LC/MS raw spectra generated in these assays.

INSTRUMENT(S): TripleTOF 5600+

ORGANISM(S): Trypanosoma Cruzi (ncbitaxon:5693)

SUBMITTER: Concepcion Judith Puerta Bula

PROVIDER: MSV000087835 | MassIVE | Fri Jul 16 07:50:00 BST 2021

SECONDARY ACCESSION(S): PXD027371

REPOSITORIES: MassIVE

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Project description:RNA pull-down assay. For the recombinant protein pull-down assays, 50 M-5g of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4M-0C. 100 M-5g of total RNA from epimastigotes were incubated with the bound protein in 500 M-5l EMSA buffer at 4M-0C for 2 h, in the presence of Heparine and Spermidine as competitors. Bounded and supernatant samples were separated. The bound sample was washed with the same buffer three times, soft-mixing for 10 min each. After washing, RNA present in the bound and supernatantM- fractions were purified. RNA purification and amplification: RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpM-^YII aRNA Amplification kit (Ambion), according to the manufacturerM-^Rs manual. Microarray analysis: The microarray was constructed with 70-mer oligonucleotides. Due to the hybrid and repetitive nature of the sequenced T. cruzi strain, all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered by the BLASTClust program, using parameters of 40% coverage and 75% identity. For probe design, it was used ArrayOligoSelector software (v. 3.8.1), with a parameter of 50% G+C content. Was obtained 10,359 probes for the longest T. cruzi CDS of each cluster, 393 probes corresponded to the genes of an external group (Cryptosporidium hominis) and 64 spots contained only spotting solution (SSC 3x), given 10,816 spots in total. These oligonucleotides were spotted from a 50 M-5M solution onto poly-L-lysine coated slides and cross-linked with 600 mJ UV. Each probe corresponding to the T. cruzi genes was identified according to the T. cruzi Genome Consortium annotation (www.genedb.org). We compared bound and unbound mRNA, extracted from two independent pull-down assays, in a dye-swap design including four slides. Microarray images were analyzed by Spot software (Spot). The Limma package (Smyth GK, 2004) was used for background correction by the normexp method, intra-slide normalization by the printtiploess method and inter-slide normalization by the quantile method. The results for the two intra-slide probe replicates were then averaged. The pull-down results were averaged, and probes displaying more than a two-fold difference between the bound and unbound fractions were selected, at FDR 1%.

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Trypanosoma cruzi proteins identified in pull-down assays aimed to characterize RNA proteins interacting with LYT1 mRNAs

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