Abstract: Small interfering RNA or silencing RNA plays a variety of roles in cellular process of organisms. Studies in bacteria and archaea had gene silencing pathways that are triggered by transgene expression or viral replication. RNA silencing mechanism is first recognized as antiviral mechanisms that protect organisms from RNA viruses, or which prevent the random integration of transposable elements. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are arrays of prokaryotic DNA sequences that mediate a form of acquired immunity to specific viral pathogens. CRISPR-associated (CAS) proteins play an important role in the initial recognition of phage genetic material and incorporating these proto-spacers in the CRISPR array. Phage-derived spacers are incorporated at the CRISPR 5' leader sequence so that the ordered sequence of spacers essentially gives a temporal record of the infection history in that bacterial population. Hence, CRISPRs offer a high-resolution method for molecular typing of bacterial hosts and their pathogens based on the unique CRISPR signature.
INTRODUCTION
Small RNAs perform diverse biological functions. They function by guiding sequence-specific gene silencing at the transcriptional and/or post-transcriptional level. The process by which double-stranded RNAs set off the degradation of homologous RNA is known as RNAi (RNA interference). Several diverse classes of small RNAs, which undergo this process, have been discovered to play key regulatory roles in diverse cellular processes (Mondal, 2003). According to their origin or function, three types of naturally occurring small RNAs have been described; short interfering RNAs (siRNAs), repeat-associated short interfering RNAs (rasiRNAs) and microRNAs (miRNAs) (Ding and Voinnet, 2007). In nature, double stranded RNA (dsRNA) can be produced by RNA-templated RNA polymerization or by hybridization of overlapping transcripts. Such dsRNAs give rise to siRNAs or rasiRNAs, which generally guide mRNA degradation and/or chromatin modification (Mojica et al., 2005). In addition, endogenous transcripts that contain complementary or near-complementary 20-50 bp inverted repeats fold back on themselves to form dsRNA hairpins. These dsRNAs are processed into miRNAs that mediate translational repression and they may also guide mRNA degradation (Mondal, 2003; Ding and Voinnet, 2007).
DISCOVERY OF RNAi PATHWAY
RNA-mediated gene silencing can be categorized into two partially overlapping pathways; RNAi pathway and miRNA pathway. Both pathways have been widely distributed in eukaryotes wherein they are served as a defense system to protect host genomes from foreign genetic elements. RNAi is triggered by either endogenous or exogenous dsRNA and silences endogenous genes carrying homologous sequences. The dsRNA molecules in RNAi pathway are processed by Dicer RNase III proteins into small RNAs, which are then loaded into silencing complexes either RNA Induced Silencing Complexes (RISC) for post-transcriptional silencing or RNA-induced initiation of transcriptional gene silencing (RITS) complexes for transcriptional silencing (Mondal, 2003; Lares et al., 2010).
CRISPR-CAS ARCHITECTURE
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are a peculiar family of DNA repeats widely distributed in the genomes of bacteria and archaea. CRISPR loci are present in ~40% of eubacterial genomes and nearly all archaeal genomes sequenced to date and consist of short (~24-48 nucleotide) repeats separated by similarly sized, unique spacers. They show characteristics of both tandem and interspaced repeats (Held et al., 2010). There is a tendency for existing CRISPRs in thermophiles more than in mesophiles. Some of the CRISPR sequences from Thermoanaerobacter tengcongensis and Thermus thermophilus HB8 are unique to the megaplasmid on which others are shared with bacterial chromosomes. Geographic distribution of spacers and CRISPR loci and their diversity within extremophiles are sometimes providing the importance to reveal its hypothetical molecular machinery reliably (Kunin et al., 2007; Diez-Villasennr et al., 2010; Deveau et al., 2010). CRISPR-CAS system has comprised the following genome components (Mojica et al., 2005; Horvath and Barrangou, 2010). They are:
Direct Repeat (DR): A CRISPR is a succession of 21-47 bp sequences called direct repeat separated by unique sequences of a similar length. The repeats are almost always identical with respect to size and sequence. Despite being divergent between species, repeats can be clustered into at least 12 major groups. Some of the larger groups contain a short (5-7 bp) palindrome which has been inferred to contribute to an RNA stem-loop secondary structure of the repeat. It can be supported both by existence of compensatory mutations in the repeats that maintain the stem structure and by observations that repeat-spacer array is transcribed into RNA. Thus, both the structural features and the conserved 3' motif GAAA(C/G) have been suggested to act as binding sites for one or more of the CRISPR-associated (CAS) proteins (Jansen et al., 2002; Mojica et al., 2005; Kunin et al., 2007).
Leader: Chromosomal clusters carry a flanking sequence at one end is referred to as a leader, although its function is unknown. The leader sequences tend to be rich in short homopolynucleotide sequences and AT-rich regions and they lack open reading frames. Archaeal flanking sequences range in size from 132 bp for Haloarcula marismortui to 564 bp for Methanopyrus kandleriand they directly adjoin the first repeat sequence of the cluster (Lillestol et al., 2006). They invariably occur at the same end of the cluster with respect to the strand orientation of the repeat sequence. There is an approximate direct correlation between the sequence length and the optimal growth temperature of the organism. A new repeat-spacer unit is almost always added to the CRISPR array between the leader and the previous unit, which suggests that the leader could function as a recognition sequence for the addition of new spacers. The leader has also been suggested to act as the promoter of the transcribed CRISPR array, as it is found directly upstream of the first repeat (Pougach et al., 2010).
Spacer: The repeated sequences, typically specific to a given CRISPR locus, are interspaced by variable sequences of constant and similar length, called spacers. The size of them is usually 20 to 58 bp depending on the species or the CRISPR locus. Spacers vary in size from 35 to 44 bp between clusters as well as between organisms (Aklujkar and Lovley, 2010; Pougach et al., 2010). All spacer sequences within a cluster and within a chromosome are generally different. Spacers are occasionally repeated, sometimes more than once within a cluster and can appear in different clusters within the same chromosome (Lillestol et al., 2006).
CAS GENE FAMILY
CAS genes are always found closely linked to the repetitive sequences. A comprehensive bioinformatics analysis of the CAS system in sequenced genomes resulted in a refined classification with 25 gene families and at least nine types of the CAS operon organization (Jansen et al., 2002) and examples of some CAS operons are depicted in Fig. 1. Eight CAS protein families have been predicted to possess nuclease activity and nine families have been characterized as putative RNA-binding proteins (RAMP domain proteins). Two families have been predicted to possess heliCASe and DNA/RNA polymerase (Horvath and Barrangou, 2010). Phylogenetic studies performed on the CAS protein family suggest that CRISPRs are acquired by horizontal transfer activity (Makarova et al., 2006; Marraffini and Sontheimer, 2009, 2010). The list of CAS proteins and corresponding molecular functions are summarized in Table 1.
CAS1 gene encodes a highly conserved protein and that is represented in all CAS neigh borhoods, with the single exception of thermophilic archaea, Pyrococcus abyssii (Makarova et al., 2006). CAS1 protein may act as a novel nuclease/ integrase. It has a metal-dependent DNase activity so that it may be involved in the initial recognition and acquisition of viral motifs. It is used as the best marker of the CRISPR-associated systems in prokaryotic genomes (Jansen et al., 2002; Deveau et al., 2010).
CAS2 is another common gene in CAS gene family, which usually located immediately downstream of CAS1 gene. The members CAS2 superfamily are small proteins with 80-120 amino acids. It has distinct structural motifs, particularly an N-terminal β-strand followed by a polar amino acid (aspartate or asparagines). CAS2 family proteins are ssRNA-specific endoribonucleases and cleave it within U-rich regions. CAS3 gene in CAS system encodes heliCASe, which is unusually long and proteins. CAS4 gene encodes a RecB family nuclease usually containing a C-terminal Zn cluster. RAMP family is belonged to CAS5 superfamily.
| Fig. 1: | Some of the CAS operon/gene clusters |
| Table 1: | Functional description of CAS protein superfamily |
CAS6 protein acts as a novel endoribonuclease. CAS6 protein interacts with a specific sequence motif in the 5’ region of the CRISPR repeat element and cleaves at a defined site within the 3’ region of the repeat and then release individual invader targeting RNAs. The functions of several other CAS gene families remain obscure (Makarova et al., 2006; Koonin and Makarova, 2009; Garneau et al., 2010).
The Dev and CAS genes in Myxococcus xanthus are co-transcribed with a short upstream gene and at least two repeats of the downstream CRISPR located at the dev locus (forming the dev operon), which is adjacent to CRISPR system (Viswanathan et al., 2007).
STRUCTURE OF RAMPS
RAMPs are the most diverse class of CAS genes which have G-rich loop at the C-terminus. The crystal structure of this protein from Thermus thermophilus reveals that the RAMP module is a duplication of a ferredoxin-like fold domain. Each domain has a two layer α+β architecture. It is composed of four β-strands and two α-helixes. It contains a small domain preceding the HD-hydrolase N-terminal domain present in many CAS3 helices topologically arranged as a repeat of two βαβ units. The N-terminal ferredoxin-like domain contains two additional α-helices inserted before and after the first α-helix. The C-terminal domain has two disordered regions and houses the conserved Gly-rich loop situated between the last α-helix and β-strand (Makarova et al., 2006).
DEFENSE MECHANISMS AGAINST FOREIGN GENETIC ELEMENTS
Genomes are potential targets of invasion by molecular parasites such as viruses and transposable elements and organisms have evolved RNA-directed defense mechanisms to cope with the constant threat of genome invaders (Barrangou et al., 2007). CRISPR-CAS systems protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. The proposed CRISPR-CAS pathway is represented in Fig. 2. Although, CRISPRs found to play a major role in replicon partitioning, DNA repair, regulation, DNA mobilization and chromosomal rearrangement. The CRISPR spacers and repeats are transcribed and processed into small CRISPR RNAs (crRNAs) that specify acquired immunity against bacteriophage infection by a mechanism that relies on the strict identity between CRISPR spacers and phage targets (Ding and Voinnet, 2007; Horvath and Barrangou, 2010; Vale and Little, 2010).
| Fig. 2: | The proposed molecular mechanism of psiRNA processing to cleave the target RNA molecule |
| psiRNA: | Prokaryotic short interfering RNA; xRISC: prokaryotic RNA induced silencing complexes |
| Step 1: | Cas proteins complexes target and cleave short recognition motifs in the phage genome and incorporate these proto-spacers into the host genome at the 5' end of the CRISPR locus. |
| Step 2: | These incorporated spacers, flanked on either side by partial repeat sequences, are transcribed as crRNAs resulting in large-scale amplification of that specific sequence. |
| Step 3: | Interference with phage-derived sequences is again mediated by Cas-protein complexes thereby crRNAs serve as templates to target conserved viral motifs in subsequent infections. |
Some spacer sequences match sequences in phage genomes and these spacers can be derived from phage and subsequently help protect the cell from infection (Levin, 2010). As a result, an active CRISPR repeat array may evolve rapidly. Repeat clusters are generally highly conserved in their repeat sequences and in the sizes of their repeats and spacers. Such integrity extends to an almost complete lack of insertion sequences or other mobile elements. Thus, the large number of mobile elements in some archaeal and bacterial genomes and the large variety of spacer sequences could provide potential target sites for the insertion of mobile elements. Many reports support the existence of an RNA-mediated genome defense pathway in archaea and numerous bacteria that has been hypothesized to parallel the eukaryotic RNAi pathway. However, these regulatory RNAs are not generally considered to be analogous to miRNAs because the dicer enzyme is not involved (Mojica et al., 2005; Ding and Voinnet, 2007; Sorek et al., 2008; Marraffini and Sontheimer, 2009; 2010).
FUTURE SCENARIO
Repetitive sequences are common in the prokaryotic genomes and their identification is increasingly facilitated by the availability of sequences of complete bacterial genomes (Chellapandi et al., 2007, 2009, 2010a). Indeed, prophages account for most of the genetic variation among closely related strains and have greatly contributed to their evolution (Chellapandi, 2010). Unlike mesophiles, thermophiles have unique molecular mechanism to uptake foreign genetic elements. However, phylogenetic analyses clearly revealed the function of identified key proteins which functions still not known in thermophiles. Phylogenetic relationship of CAS protein family in thermophilic bacteria was absolutely varied from thermophilic archaea. The length, sequence and position of the sequences of the protein families in archaeal genome are highly variable and often unique for a single strain (Karthigeyan et al., 2007; Vedhagiri et al., 2009; Chellapandi et al., 2007, 2009, 2010a). Hence, unique extremophilic adaptations to drastically varying biosystems have aroused special interests in their respective potential in biotechnological applications. Available genome sequences of thermophiles and bioinformatics databases, resources and software tools provide an importance for revealing the molecular hypothesis on CRISPR-CAS system and metabolomes of prokaryotes (Razia et al., 2010; Chellapandi and Kalaimathy, 2010; Chellapandi and Dhivya, 2010; Chellapandi et al., 2010b).
APPLICATION OF CRISPR-CAS SYSTEM
CRISPR-CAS system has become a widely used tool to knock down and analyze the function of genes, especially in non-model organisms where the systematic recovery of mutants is not feasible. The potential for using this mechanism is to identify new drug targets and for designing small molecule drugs. Perhaps, this system would be useful for strain typing of pathogenic bacteria, engineered defense against virus in industrial bacteria, selective silencing of endogenous genes for revolutionize microbial-physiology research by CRISPR arrays (Spoligotyping) (Makarova et al., 2006; Sorek et al., 2008; Cadmus et al., 2010).
GENERAL DEFINITION RELATED TO CRISPR-CAS SYSTEM
| • | Spoligotyping: A technique is used to differentiate different strains of the same species according to differences in the spacers in their CRISPR arrays |
| • | MiRNAs: Genomically encoded non-coding RNAs that help regulate gene expression, particularly during development |
| • | RISC: A multiprotein complex that incorporates one strand of a siRNA or miRNA |
| • | RITS: A form of RNA interference by which siRNA - trigger the downregulation of transcription of a particular gene or genomic region. |
| • | Dicer: An endoribonuclease in the RNase III family that cleaves dsRNA and pre-microRNA into short dsRNA fragments about 20-25 nucleotides long, usually with a two-base overhang on the 3′ end. |
| • | RAMP: A cytoplasmic and nuclear protein that associates with double-strand or single-strand RNAs through RNA recognition motif. |
| • | RasiRNA: A subclass of Piwi-interacting RNAs (piRNAs), which are small RNA molecule which interact with Piwi proteins |
| • | Horizontal gene transfer: Any process in which a bacterium incorporates genetic material from distantly-related bacteria without being the offspring of that organism |