Identification of Alternative Splicing Events Associated with Paratuberculosis in Dairy Cattle Using Multi-Tissue RNA Sequencing Data

Abstract

Paratuberculosis is a major endemic disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection and leads to huge economic loss in the dairy sector worldwide. Alternative splicing (AS) events, playing indispensable regulatory roles in many protein functions and biological pathways, are shown to be associated with complex traits and diseases. In this study, by integrating the RNA sequencing (RNA-seq) data of 24 samples from three tissues (peripheral blood, jejunum and salivary gland) of Holstein cows, we obtained 2,706,541,696 uniquely mapped reads in total that represented 12,870 expressed genes, and detected 4285 differentially expressed genes (DEGs) between MAP-infected and healthy cows (p < 0.05). Of them, 92 differentially expressed splicing factors (DESFs) were included. Further, 119, 150 and 68 differential alternative splicing (DAS) events between MAP-infected and healthy cows were identified in peripheral blood, jejunum and salivary glands, respectively. Of note, six DAS events were highly and significantly correlated with the DESFs (R2 > 0.9; p < 0.01), and their corresponding genes (COPI coat complex subunit gamma 2gene (COPG2), kinesin family member 2C gene (KIF2C), exocyst complex component 7 (EXOC7), Rab9 effector protein with kelch motifs gene (RABEPK), deoxyribonuclease 1 gene (DNASE1) and early endosome antigen 1gene (EEA1)) were significantly enriched in immune response such as vesicle-mediated transport, regulation of acute inflammatory response and tuberculosis through gene ontology (GO) and KEGG analysis. KS test showed that the DAS events in the EXOC7 and KIF2C genes indeed displayed differences between MAP-infected cows and healthy cows. The DAS in EXOC7 might produce a new protein sequence with lack of 23 amino acids, and the DAS in KIF2C induced a stop codon of premature occurrence and resulted in a lack of functional domain. In summary, this study identified the DAS events and corresponding genes related to MAP-infection base on the RNA-seq data from multiple tissues of Holstein cows, providing novel insights into the regulatory mechanisms underpinning paratuberculosis in dairy cattle.

Comparative proteome analysis revealed the differences in response to both Mycobacterium tuberculosis and Mycobacterium bovis infection of bovine alveolar macrophages

“…This mechanism might contribute to bacteria evasion from the host macrophage immune responses. Hosts exhibit varied gene regulatory responses to MTB infection, encompassing mechanisms such as long noncoding RNAs (lncRNAs), miRNAs, and splicing events (Kumar et al, 2016;Pawar et al, 2016;Li et al, 2022). Notably, both lncRNAs and miRNAs influence autophagy-related signalling pathways, serving as pivotal players in modulating MTB immunity (Ouimet et al, 2016;Zhang et al, 2022).…”

Characterization of peripheral white blood cells transcriptome to unravel the regulatory signatures of bovine subclinical mastitis resistance

“…These AS events are strongly associated with disease resistance in cows. For example, two differential alternative splicing (DAS) events in the genes EXOC7 and KIF2C affect protein functional domains and are associated with the susceptibility to Mycobacterium avium subspecies paratuberculosis ( Li et al, 2022 ). Furthermore, the specific alternative splicing patterns exhibited in genes SLAMF7 ( Ju et al, 2012a ), BOLA-DQA2 ( Hou et al, 2012 ), and CD46 ( Wang et al, 2016 ) in mammary gland tissues of cows were associated with mastitis resistance in cows.…”

Immunomodulatory effects of vitamin D3 on dairy cattle naturally infected with Mycobacterium avium subspecies paratuberculosis

“…tb can also disrupt endosomal trafficking and lysosomal maturation through alternatively splicing of GTPase Rab8B (RAB8B) transcripts in human MDMs resulting in ineffective protein (96). MAP has also been shown to utilize alternative splicing events in bovine paratuberculosis targeting vesicle trafficking, macrophage activation, and lysosomal function (97,98).…”