Mechanism of RAG Regulation During Its Physiological and Pathological Functions in Lymphoid Cells

Abstract

RAGs (Recombination Activating Genes) are responsible for generation of antigen receptor diversity in case of B-cells and T-cells, through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. Each of these segments are flanked by recombination signal sequences (RSS), which consist of a conserved heptamer and nonamer separated by a less conserved spacer of 12 or 23 bp. RAGs recognize and cleave at the 5’ end of heptamer, leading to the formation of hairpin coding ends and blunt signal ends. The coding ends are joined through the process of no homologous DNA end joining (NHEJ), leading to the rearrangement of variable region of antigen receptors. Apart from its physiological property, RAGs can also act as a structure-specific nuclease. Previously, it has been shown that inadvertent action of RAGs on cryptic RSS and non B-DNA structures can lead to the generation of genomic instability and cancer. A very coordinated expression of RAGs has been observed in pro- and pre-B cells of the lymphoid system, which overlaps with the window of productive rearrangement during V(D)J recombination. Besides, studies by us and others have shown that RAG cleavage at altered DNA structures and cryptic RSS leads to chromosomal translocations resulting into cancer. However, several questions related to regulation of RAG expression and its activity in lymphoid cells remains to be answered. Previous studies have suggested regulation of RAG expression at different levels, such as methylation, ubiquitination, phosphorylation and by coordinate action of various transcription factors. In the present study, we evaluate the potential role of miRNAs in the regulation of RAG expression and its function in lymphoid cells. miRNAs are small, single-stranded non-coding RNAs, which play an important role in the regulation of gene expression. They play a critical role in the regulation of different cellular functions. Although there are miRNAs identified to play critical role during development of immune system, several key questions such as its role in the regulation of RAGs is yet to be addressed. In the current study, we have used bioinformatics approach to extract potential miRNAs that bind to 3’UTR of RAG1 and RAG2. miRNA expression datasets were downloaded from NCBI SRA database and extensive evaluation was done using various bioinformatics tools such as Bowtie, Sam tools, Bam tools, Bed tools and R package. We screened the miRNA expression profile across different stages of B-cell development (pro, pre, immature and mature B-cells), which overlap with the narrow window of RAG expression. The shortlisted miRNAs were further analyzed using miRNA databases such as miRBase, Targetscan and EMBL. Results showed that 33 miRNAs were specific to RAG1, among that one (miRNA1) followed RAG expression profile in B-cells. Besides miRNA2, which is a novel miRNA, was selected only on the basis of RAGs expression profile in a stage specific manner and the complementarity of the seed sequence of miRNA2 to the 3’UTR of RAG1 was checked manually. Interestingly, we observed that RAG1 expression was significantly down regulated in the presence of these miRNAs. However, there was no significant difference in the levels of other genes analysed. Further, semi-quantitative RT-PCR analysis confirmed the endogenous processing of pre-miRNA into mature miRNA using the cellular machinery. Besides, enrichment of 3’UTR of seed region of these miRNAs, enhanced the expression level of RAG1. Importantly, the enhancement in RAG1 expression level was limited in case of mature B-cells, where RAG expression is normally not observed. Further, transfection of lymphoid cells with miRNA inhibitors, specific to the miRNAs under study, showed the enhancement in RAG1 expression in lymphoid cells. In addition to this, specificity of selected miRNAs was confirmed by performing 3’UTR reporter assays, where enhanced luciferase expression was observed in case of mutant 3’UTR, while it was minimal in case of wild type constructs. Endogenous expression levels of selected miRNAs were evaluated in both lymphoid and nonlymphoid normal tissues and cancer cells using RT-PCR. Interestingly, we observed inverse correlation of expression levels of miRNA and RAG expression in all the cells tested. Besides, miRNA expression levels were less in pre-B cells and T-cells, owing to the increased expression of RAGs. Apart from this, recombinogenic potential of candidate miRNAs was assessed using episomal based V(D)J recombination assays. Interestingly we observed significant decrease (2-4 fold) in the V(D)J recombination efficiency when miRNA1 or 2 constructs were transfected in Nalm6 cells, as compared to that of controls, where no miRNAs were used. However, in case of Reh cells upon transfection with miRNA1construct, the decrease in recombination potential was upto 9 fold. Hence, we identify two miRNAs that can play an important role in the regulation of RAG1 expression and its physiological activity. Further, studies are being carried out to confirm their role in the regulation of RAG1 during different developmental stages of lymphoid cells in mice. As stated above, in addition to the sequence-specific activity, RAG possesses structure-specific nuclease activity as well. It has been shown that RAGs can cleave different types of altered DNA structures. Studies from our laboratory showed that even when RAGs act as a structure-specific nuclease there is a sequence bias. Presence of cytosine and thymine at the single-stranded region of heteroduplex DNA is important for RAG nicking and double-strand break (DSB) formation. In addition, proximity of a nonamer to bubble structures can enhance RAG cleavage. However, the role of immediate flanking sequences in the RAG mediated cleavage at heteroduplex regions is not understood. We investigated the role of flanking double-stranded DNA sequences in the regulation of RAG cleavage on non-B DNA structures. We found that RAG binding and cleavage on heteroduplex DNA is dependent on the length of double-stranded flanking region. Besides, immediate flanking regions of the heteroduplex DNA affected the RAG binding and cleavage in a sequence dependent manner. Interestingly, we also observed that the cleavage efficiency of RAGs at heteroduplex region was influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights into regulation of the pathological action of RAGs. Previous studies have shown that in addition to formation of coding and signal joints during V(D)J recombination, nonstandard V(D)J recombination products known as hybrid joints and open-shut joints may be formed, particularly in certain aberrant conditions such as defective NHEJ machinery. Interestingly, the hybrid and open-shut joints closely resemble the transposition mechanisms associated with transposons oretroviruses. Studies have also shown that RAGs possess structural similarity with integrases in domain organization. Both the proteins have Zinc Finger Binding domain (ZFB) which helps in multimerization of the protein, a central catalytic core domain comprising three acidic amino acids D, D and E essential for enzymatic activity and C-terminal domain (CTD) responsible for nonspecific binding to the DNA. Previous studies from our laboratory showed that, Elvitegravir, an inhibitor of integrase could interfere with the biochemical functions of the RAGs in vitro. Specifically, it inhibited the RAG binding and cleavage at RSS, hairpin formation, post-cleavage complex formation involving 12RSS and 23RSS. Using the episomal assay system that mimics signal joints (pGG49) and coding joints (pGG51), we show that Elvitegravir can inhibit V(D)J recombination inside cells. Interestingly we observed 3-6 fold decrease in the recombination frequency in signal ends joining, when treated with increasing concentrations (100, 500 and 1000 nM) of Elvitegravir. A 5-8 fold decrease in coding joints formation was also observed upon treatment with the inhibitor. The presence of recombination was confirmed by restriction digestion followed by sequencing analysis. Further analysis of recombination junctions revealed extensive deletion before joining in the case of Elvitegravir treated samples. Insertions or substitutions near to the recombination junctions were also prominent in treated samples. In depth analysis of sequenced junctions showed the presence of sequence having the features to form hairpins both upstream and downstream to the RSS sequences and was the site of cleavage in cases were higher deletion was observed. The analyzed recombinants did not show any signal joints or coding joints formation in treated samples. This suggests that Elvitegravir affects the physiological function, the V(D)J recombination of RAGs inside the cells. Thus, in the present study, we show that RAGs can be regulated by specific miRNAs. We have identified two potential miRNAs, which can regulate the RAG expression as well as its function in different stages of B- and T-cell development. Further, we also identify a novel regulatory mechanism for the structure-specific activity of the RAG complex. In addition to this, we find that integrase inhibitor, Elvitegravir, affects V(D)J recombination within B-cells, indicating its potential deleterious impact in HIV patients, which needs to be further evaluated.