Dataset Information

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.

SUBMITTER: Kalam H

PROVIDER: S-EPMC7115969 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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

Kalam Haroon H Singh Kartikeya K Chauhan Komal K Fontana Mary F MF Kumar Dhiraj D

IUBMB life 20180818 9

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 impa ...[more]

PMID: 30120868

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Project description:Transcriptional reprogramming of macrophages upon Mycobacterium tuberculosis (Mtb) infection is widely studied; however, the significance of alternate splicing (AS) in shaping cellular responses to mycobacterial infections is not yet appreciated. Alternate splicing can influence transcript stability or structure, function and localization of corresponding proteins thereby altering protein stoichiometry and physiological consequences. Using comprehensive analysis of a time-series RNA-seq data obtained from human macrophages infected with virulent or avirulent strains of Mtb, we show extensive remodeling of alternate splicing in macrophage transcriptome. The global nature of this regulation was evident since genes belonging to functional classes like trafficking, immune response, autophagy, redox and metabolism showed marked departure in the pattern of splicing in the infected macrophages. The systemic perturbation of splicing machinery in the infected macrophages was apparent as genes involved at different stages of spliceosome assembly were also regulated at the splicing level. Curiously there was a considerable increase in the expression of truncated/non-translatable variants of several genes, specifically upon virulent infections. Increased expression of truncated transcripts correlated with a decline in the corresponding protein levels. We verified the physiological relevance for one such candidate gene RAB8B; whose truncated variant gets enriched in H37Rv infected cells. Upon tweaking relative abundance of longer or shorter variants of RAB8B transcripts by specialized transduction, mycobacterial targeting to lysosomes could be promoted or blocked respectively, which also resulted in corresponding changes in the bacterial survival. Our results show RAB8B recruitment to the mycobacterial phagosomes is required for phagosome maturation. Thus the abundance of truncated RAB8B variant helps virulent Mtb survival by limiting the RAB8B levels in the cells, a mechanism which we subsequently verified in human primary macrophages. Taken together we demonstrate alternate splicing as a new locus of intervention by Mtb and provide attractive alternative to exploit for novel drug targets against Mtb.

Project description:Genome-scale metabolic models of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, have been envisioned as a platform for drug discovery. By systematically probing the networks that underpin such models, the reactions that are essential for Mtb are identified. A majority of these reactions are catalyzed by enzymes and thus represent candidate drug targets to fight an Mtb infection. Nevertheless, this is complicated by the limited knowledge on the environment that Mtb encounters during infection. Modeling the behavior of the bacteria during infection requires knowledge of the so-called biomass reaction that represents bacterial biomass composition. This composition varies in different environments or bacterial growth phases. Accurate modeling of the metabolic state requires a precise biomass reaction for the described condition. In recent years, additional insights in the in-host environment occupied by Mtb have been gained as transcript abundance data of interacting host and pathogen have become available. Therefore, we used transcript abundance data and developed a straightforward and systematic method to obtain a condition-specific biomass reaction for Mtb during in vitro growth and during infection of its host. The method described herein is virtually free of any pre-set assumptions on uptake rates of nutrients, making it suitable for exploring environments with limited accessibility. The condition-specific biomass reaction represents the "metabolic objective" of Mtb in a given environment (in-host growth and growth on defined medium) at a specific time point, and as such allows modeling the bacterial metabolic state in these environments. Five different biomass reactions were used to predict nutrient uptake rates and gene essentiality. Predictions were subsequently compared to available experimental data. Our results show that nutrient uptake can accurately be predicted. Gene essentiality can also be predicted but accurate predictions remain difficult to obtain. In conclusion, a viable strategy to model Mtb metabolism in hard-to-access environments that is virtually free of pre-set assumptions is provided.

Dataset Information

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

Publications

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

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