Quantitative Mass Spectrometric Analysis of RNA-Protein Cross-Links

In recent times much emphasis has been laid on revealing the composition and regulation of various RNP complexes. The structural studies of the RNP complexes provide a valuable insight into the binding modes and functional implications of interactions between the RNAs and RNA binding proteins within the complexes. To investigate the interactions of the RNA-binding proteins within RNP complexes, UV-induced cross-linking followed by mass spectrometry (MS) has proved to be a promising and straightforward technique. But the limitations of most of the purification methods as well as the intricate mass spectrometric data analysis have hampered the study of these RNP complexes. During the course of this study, the protocol was modified and optimized for the interaction analysis of large RNP complex assemblies like RNP complexes isolated from the HeLa nuclear extract which led to the identification of predicted as well as unknown RBMs. Moreover, the qualitative analysis of the protein-RNA cross-links derived from in vitro assembled Brat-NHL-hb RNA complex and CWC2-U6/U4 snRNAs complexes was also carried out. Based on the qualitative analysis of CWC2-U6/U4 cross-links, the quantitative analysis of protein-RNA cross-links has been established. The studies conducted during the research work have contributed in the identification and characterization of protein-RNA interactions within the aforementioned complexes and also provided the quantitative insight into the protein-RNA interactions.2020-03-0

Identification of targets of Human Cytomegalovirus microRNAs by cross linking and analysis of cDNA (CRAC).

The discovery of a class of small ribonucleic acid (RNA) molecules known as microRNAs (miRNAs) has led to extensive interest in their biological relevance and role. The first miRNA was discovered in Caenorhabditis elegans in 1993. Since then studies have shown that miRNAs represent a fundamental mechanism of gene expression regulation, regulating thousands of genes at the post-transcriptional level. Given that viruses are highly adept at exploiting cellular processes, it is perhaps unsurprising they have evolved miRNAs of their own. The majority of known viral miRNAs are expressed by herpes viruses underscoring their importance to this virus family. Identifying the targets of herpes virus miRNAs would aid in elucidating the role played during infection. In this study, we aim to identify and understand the targets of the human cytomegalovirus (HCMV) encoded miRNAs using a cutting edge biochemical technique, Cross-Linking and Analysis of cDNA (CRAC). HCMV is a member of the beta (β) herpes virus subfamily and is globally distributed causing clinically asymptotic infections in immune competent individuals. However, persistent and recurrent infections in AIDS and organ transplant patients, who have a compromised immune function causes a high degree of mortality and morbidity. Intra-uterine infected infants are also at high risk with infection causing congenital abnormalities and mental retardation. Scientists worldwide are trying to understand one special characteristic of HCMV and herpes viruses in general, which is latency i.e. the presence of an intact viral genome in the cell with a majority of the genes in a dormant or silent state. Viral encoded miRNAs regulate both the viral genome as well as the host’s and this has been postulated as a latency inducing mechanism. To date, there are 22 known HCMV encoded miRNAs. On-going research in our group using techniques such as bioinformatics, RISC immunoprecipitation (RISC-IP) and microarray analysis has identified both viral and cellular targets of HCMV miRNAs. Targets include a crucial viral transactivator thought to play an important role in latency as well as cellular targets involved in a variety of functions including cell cycle control, intrinsic defence and innate cellular defence. However, the majority of HCMV miRNAs have no known function. New approaches and technologies are required to elucidate the functions of these miRNAs. The initial goal of this project is to establish and optimise the CRAC technique. This will be performed in HEK293 cells transfected with a plasmid expressing a cluster of HCMV miRNAs termed miR-US25. The long-term goal of the project will be to use CRAC technology to identify miRNA targets in infected cells

Systematic analysis of host-cell interactions during human cytomegalovirus infection

Viruses are obligate intracellular pathogens. Therefore, their successful replication, at every stage from attachment to assembly and egress, is dependent on host cell functions. The host cell in turn engages mechanisms to counteract virus replication. As a result, viruses have evolved mechanisms to evade these counteracting measures as well as ways to reshape the cellular environment into one that’s favourable for successful replication. Systematic studies offer a platform for unravelling virus-cell interactions and in particular can address three important aspects 1) increase our understanding of basic biology of the virus, 2) identify and characterise novel cellular functions 3) provide important leads for novel targets for antiviral therapy. In this study, I investigated two aspects of virus host interaction; the role of microRNAs (miRNAs) in virus infection and the role of interferon inducible genes in virus infection. Human cytomegalovirus (HCMV) is a β herpes virus that infects humans. HCMV maintains a persistent lifelong infection in the host involving a cycle of latency and reactivation. Infection of healthy individuals with HCMV results in relatively minor symptoms. In contrast, infection of individuals with a compromised immune system, as in the case of organ transplant recipients and AIDS patients, can cause significant morbidity and mortality. In common with other herpes viruses, HCMV expresses multiple small regulatory RNAs called miRNAs. HCMV encodes at least 14 miRNAs. Identifying the targets of these miRNAs will help us understand their functional importance during infection. Recently, a biochemical technique called Cross-Linking, Ligation and Sequencing of Hybrids (CLASH), was developed by Tollervey and colleagues, representing the most advanced systematic technique for the identification of miRNA targets. We adapted this approach to identify high confidence miRNA targets during HCMV infection. However, the protocol was sub-optimal and presented us with technical challenges. Although high quality data sets were not generated, the work was crucial for the establishment of the system which is now generating promising data. Virus-cell interactions can also be elucidated by probing for host factors that are important for virus replication. Type I interferon is a highly effective inhibitor of HCMV replication. Treatment of cells with interferon results in up regulation of multiple effectors known as interferon stimulated genes (ISGs). How these genes block HCMV replication is poorly understood. A library of more than 380 ISG expressing lentiviruses was screened to determine the effects of individual ISGs on HCMV replication. The screen was performed in primary human fibroblast cells and a glioblastoma cell line called U373s. Multiple inhibitory ISGs were identified including well characterised ISGs such as cGAS, STAT2, NOD2, DDX60 and HPSE as well as novel candidates TXNIP, ELF1, FAM46C, MT1H and CHMP5. Five ISGs were identified as HCMV replication enhancers including previously published ISGs BST2 and IFITM1 and novel enhancers ODC1, BCL3 and IL28RA. siRNA screens against top hits demonstrated that STAT2, CPT1A and cGAS are dominant inhibitory factors during HCMV infection and knockdown of these genes can partially rescue HCMV replication following interferon treatment. Finally, using a corresponding rhesus ISG library we show that rhesus SAMHD1 effectively inhibits HCMV replication while human SAMHD1 has no effect, suggesting that HCMV expresses a species-specific inhibitor of SAMHD1. This study defines interferon stimulated pathways important for HCMV replication and identifies multiple novel host factors that both restrict and enhance HCMV replication. These studies demonstrate the effectiveness of using systematic approaches for the identification of novel host virus interactions