Alternate splicing of transcripts upon Mycobacterium tuberculosis infection impacts the expression of functional protein domains

Abstract

Previously, we reported that infection of human macrophages with Mycobacterium tuberculosis (Mtb) results in massive alterations in the pattern of RNA splicing in the host. The finding gained significance since alternate spliced variants of a same gene may have substantially different structure, function, stability, interaction partners, localization, and so forth, owing to inclusion or exclusion of specific exons. To establish a proof-of-concept; on how infection-induced RNA splicing could impact protein functions, here we used RNA-seq data from THP-1 macrophages that were infected with clinical isolate of Mtb. In addition to re-establishing the fact that Mtb infection may cause strain specific alterations in RNA splicing, we also developed a new analysis pipeline resulting in characterization of domain maps of the transcriptome post-infection. For the sake of simplicity, we restricted our analysis to all the kinases in the human genome and considered only pfam classified protein domains and checked their frequency of inclusion or exclusion due to alternate splicing across the conditions and time points. We report massive alterations in the domain architecture of most regulated proteins across the entire kinases highlighting the physiological importance of such an understanding. This study paves way for more detailed analysis of different functional classes of proteins and perturbations to their domain architecture as a consequence of mycobacterial infections. Such analysis would yield unprecedented depth to our understanding of host-pathogen interaction and allow in a more systematic manner targeting of host pathways for controlling the infections. © 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(9):845-854, 2018.

Keywords: JAL2287; RNA-seq; alternative splicing; kinase domain; mycobacterium; pfam.

FIG 1

RNA-seq analysis of THP-1 macrophages infected with avirulent, virulent or clinical isolate of Mycobacterium tuberculosis. A. Flow-chart of the RNA-seq experiment. THP-1 macrophages were infected with H37Ra, H37Rv, or JAL2287 for different time points, total host RNA was isolated and processed for RNA-sequencing.B. Comparative table for differentially expressed genes between H37Ra, H37Rv, and JAL2287 infected macrophages at gene level quantification (up: two fold increase in expression; down: two fold decrease in expression; UN: no change in expression)C. Global gene expression profile of THP-1 macrophage cells upon infection with H37Ra, H37Rv, or JAL2287 at 0, 6, 12, 24, 36, and 48 h post infection.

FIG 2

Gene Ontology enrichment analysis of differentially expressed genes in H37Ra, H37Rv, or JAL2287 infected THP-1 macrophages. Significantly enriched gene ontology classes (P-value < 0.001) detected at all-time points were manually classified into seven categories: cell cycle, trafficking, inflammation, metabolism, signaling, immune response and transport. Color of the circle represents a particular class and size represents the cumulative enrichment score.

FIG 3

Estimation of alternate splicing in JAL2287 infected THP-1 macrophages.A. Dot plots for isoform specific psi scores between JAL2287 infected macrophage versus uninfected control plotted for each time point. Each dot represents a single transcript. The dotted lines mark the regions beyond which transcripts had higher psi-score in JAL2287 infected cells by 0.5 or more with respect to uninfected cells (red) or in uninfected cells by 0.5 or more with respect to JAL2287 infected cells (blue). B. Table showing number of significant alternate splicing events where psi-score compared to the uninfected control was higher than 0.5 across (i) JAL2287 (ii) H37Rv, (iii) H37Ra across all time points is shown here. Numbers for H37Rv and H37Ra are reproduced from Kalam et al (25) for comparative analysis. A3SS: alternate 30 splice site, A5SS: alternate 50 splice site, MXE: mutually exclusive exons, RI: retained introns, SE: skipped exons

FIG 4

Comparative domain distribution between maximally upregulated versus all isoform in JAL2287 infected THP-1 macrophage. A. Using interpro signatures domains were identified for each transcript. Frequency of pfam domains was calculated in maximally upregulated isoform per gene and compared with pfam domain frequency when all the expressed isoforms were considered. In all isoform condition a single gene will have multiple isoform while in maximally upregulated case there is only one isoform per gene. These domains frequency were used to calculate the rank in respective class as shown here.B. Dot plot of rank of domains in all isoform (Y-axis) versus rank in maximally upregulated isoform (X-axis) case. The size of the dot represents the frequency of that domain in JAL2287 infected sample 48hr post infection.C. List of domains represented in Fig. 4B.