Dataset Information

Entamoeba histolytica splicing/mRNP proteome

ABSTRACT: First, the snRNP component U1A was HA-tagged and the intron of the RabX13 gene was MS2-tagged and amoeba transformats were established with such constructs. Next, amoeba transformants were UV cross-linked and their nucler extracts were immunoprecipitated (IP) with anti-HA agarose or MS2-spharose beads. Precipitates were subjected to tandem mass spectrometry (MS/MS) analyses. MS2-IP helped to discriminate the nuclear roles (chromatin-, co-transcriptional-, splicing-related), of the proteins probed.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Entamoeba Histolytica

SUBMITTER: Robert Winkler

LAB HEAD: Dr. Jesus Valdes Flores

PROVIDER: PXD001080 | Pride | 2014-09-11

REPOSITORIES: Pride

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Publications

Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing complexes.

Valdés Jesús J Nozaki Tomoyoshi T Sato Emi E Chiba Yoko Y Nakada-Tsukui Kumiko K Villegas-Sepúlveda Nicolás N Winkler Robert R Azuara-Liceaga Elisa E Mendoza-Figueroa María Saraí MS Watanabe Natsuki N Santos Herbert J HJ Saito-Nakano Yumiko Y Galindo-Rosales José Manuel JM

Journal of proteomics 20140808

The genome of the human intestinal parasite Entamoeba histolytica contains nearly 3000 introns and bioinformatic predictions indicate that major and minor spliceosomes occur in Entamoeba. However, except for the U2-, U4-, U5- and U6 snRNAs, no other splicing factor has been cloned and characterized. Here, we HA-tagged cloned the snRNP component U1A and assessed its expression and nuclear localization. Because the snRNP-free U1A form interacts with polyadenylate-binding protein, HA-U1A immunoprec ...[more]

PMID: 25109466

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Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. The genome sequences associated with M. smegmatis CarD, RNAPb, and RNAPs were determined by ChIP-seq analysis. Samples were done in duplicate, except for RNAPs. And sequencing was performed using a SOLiD sequencer (Life Technologies).

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Dataset Information

Entamoeba histolytica splicing/mRNP proteome

Publications

Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing complexes.

Similar Datasets

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