Identification and Characterisation of a miRNA releasing activity from Caenorhabditis elegans
MicroRNAs (miRNA) are endogenous, small non coding RNAs, which play a prominent role in eukaryotic gene regulation. Perturbations leading to an altered abundance of miRNAs can lead to pathological conditions like neurological disorders, auto-immune diseases, and even cancer. Thus regulating the regulators is of utmost importance. While miRNA biogenesis is well delineated pathway, currently sparse information is available about active miRNA turnover. A mature miRNA is thought to be formed within the pre-miRISC (RNA induced silencing complex), where one of the strands, miRNA/ guide strand from the miRNA: miRNA*/ guide: passenger strand duplex, is preferentially selected and loaded onto the Argonaute (AGO). This miRNA acts as an ‘address label’, and guides the miRISC to cognate mRNA targets, and repress their expression. Researchers while purifying human siRISC have found that the small RNA remains very strongly bound by AGO, even under highly stringent conditions applied during the purification. The notion got strengthened when the crystal structure of Thermus thermophilus AGO bound to a 5’ phosphorylated guide DNA revealed that AGO has a bi-lobed structure comprising of four domains (N, PAZ, MID, and PIWI), where except the nucleotides in the seed region (2nd-8th nucleotide from the 5’ end), the entire guide sequence remains buried inside a channel. The co-crystal structure also showed that the 5’ nucleotide of the guide sequence remains anchored at the basic pocket of the MID domain, and 3’ end is bound by the PAZ domain. Later, co-crystal structures of human Ago2 with endogenous RNA, and that of yeast AGO with guide RNA, revealed that multiple general and specific contacts exist between the residues of AGO and the guide sequence. This endorsed the earlier notion that only seed sequence of the guide strand remains free for interaction with cognate targets through antisense mechanism. Together all these findings contributed to the notion that miRISC (as well as siRISC) is a super-stable structure, and the guide sequence remains inaccessible to the nucleases in the cell. Hence, miRNAs might undergo passive degradation with host AGO protein. Interestingly, this notion got upturned, when researchers working with Caenorhabditis elegans (C. elegans) demonstrated through explicit biochemical assays that miRNAs do get released from AGO, before they undergo degradation by bona fide miRNase (XRN-2), without affecting the integrity of the AGO protein. The researchers also showed that XRN-2 depleted worm lysate exhibits compromised miRNA release activity. This could possibly be because of a direct role of XRN-2 in miRNA release. Conversely, XRN-2 might function downstream of a dedicated miRNA release factor, and in XRN-2 depleted state, as the release and turnover kinetics are inter-linked, the overall miRNA release activity got diminished. However, the identity of this endogenous, and dedicated ‘miRNA release factor’ has remained elusive, so far. Here, we report, that a small 26 kDa pur-alpha family protein (PLP-1) is responsible for release of a subset of miRNAs from miRNA AGO of C. elegans, without affecting the integrity of AGO. The protein is not only capable of freeing the miRNA from the grasp of AGO, but can also bind, and deliver it to a defined component of miRNA turnover machinery (miRNasome-1). We have also found that this protein can oligomerize, and that binding of specific miRNAs can induce unique pattern of oligomerization of the protein (by enhancing specific oligomeric forms over others). This work unfurls the potential of a single protein to perform multiple functions through its different oligomeric forms, and connect miRISC/AGO to a miRNA turnover complex (miRNAsome-1) in a two-step miRNA turnover pathway. The Chapter 1 forms the Introduction to the thesis, and presents extensive literature survey on topics pertaining to the current work. The chapter begins with a brief history of non-coding RNAs, miRNAs in particular. Next, an account of the different small non-coding RNAs is presented. This is followed by a description of the well-delineated miRNA biogenesis pathway, with special emphasis on RISC (RNA induced silencing complex) and AGO proteins, the core of RISC. Next, a short summary on RNAi is documented. Further, an account of miRNA functions in general, miRNA stability, and active miRNA turnover is presented chronologically. Thereafter, a brief description of the model organism C. elegans, used for the current study is presented. The Chapter 2 constitutes the first data chapter titled ‘Identification of a miRNA releasing activity in Caenorhabditis elegans.’ This chapter embodies the primary working hypothesis, and establishes the platform for the identification of the miRNA release factor. Here an account of the step-wise fractionation of C. elegans total lysate, yielding a fraction enriched in miRNA release activity is presented. After subsequent biochemical assays with the enriched fraction, mass spectrometric analysis is carried out, which reveals Pur alpha like protein-1 (PLP-1), as a putative miRNA release factor. Using recombinant PLP-1 in in vitro assays, the protein is validated as the miRNA release factor, which can dislodge the miRNA from the grasp of AGO/ miRISC, in C. elegans. Further, a probable role of the paralogous protein PLP-2 from C. elegans in miRNA binding and release, is studied and discussed. The Chapter 3 titled ‘in vivo effects upon plp-1 knockdown in C. elegans’ follows the PLP-1 identification chapter. In this section, the consequent in vivo effects upon plp-1 knockdown is depicted. Effects on worms, both phenotypic and developmental are accounted for briefly. Next, effect on endogenous miRNAs (precursor and mature forms), and their cognate targets is assessed. This is followed by illustrating effects on other major players of miRNA metabolism namely miRNA AGO, and miRNases: XRN-1 and XRN-2, in PLP-1 depleted condition. The Chapter 4 titled ‘Functional characterization of PLP-1’, encompasses the brunt of the work, where through in vitro assays, three major functional aspects of the protein PLP-1: miRNA binding, miRNA release, and delivery of miRNA to bona fide miRNA turnover machinery, are elucidated. While demonstrating the miRNA binding property of the protein, an intriguing observation led to the revelation that the protein possess an exceptional oligomerization property. Together with biochemical assays and mutational studies, the importance of the different regions and residues of the protein, for individual functions is depicted. This chapter ends with summarising the independence or inter-dependence of the different functionalities of the protein. The thesis is concluded with a combined Discussion and Future Perspective section. This section gives a holistic picture of the work with respect to the existing knowledge on miRNA metabolism, and how the current study enhances our understanding of this process leading to the next level of perception. The following chapter contains the Materials and Methods, used in the present study. In this chapter, a comprehensive description of all the methodologies employed in the experiments is provided. References for already established methodologies are indicated and incorporated. As an addendum Appendix is included where detailed information on the different clones used in the study is provided. Also, figures corresponding to some of the data not shown in the previous version of the thesis is incorporated in this section. Finally, the closure is done with the Bibliography section.