Effect of Non-Coding RNA on Post-Transcriptional Gene Silencing of Alzheimer Disease

A large amount of hidden biological information is contained in the human genome, which is not expressed or revealed in the form of proteins; the usual end product form of gene expression. Instead, most of such information is in the form of non-coding RNAs (ncRNAs). ncRNAs correspond to genes that are transcribed, but do not get translated into proteins. This part of the genome was, till recently, considered as ‘junk’. The term ‘junk’ implied lack of any discernible function of these RNA. More than 98% of the human genomic size encompasses these non-coding RNAs. But, recent research has evidently brought out the indispensible contribution of non-coding RNA in controlling and regulating gene expression. ncRNA such as siRNAs and microRNAs have been reported to greatly help in causing post-transcriptional gene silencing (PTGS) in cells through RNA interference (RNAi) pathway. In this work, we have investigated the possibility of using siRNAs and microRNAs to aid in gene silencing of early onset Alzheimer’s disease genes. 
Alzheimer’s disease specific mutations and their corresponding positions in mRNA have been identified for six genes; Presenilin-1, Presenilin-2, APP (amyloid beta precursor protein), APBB3, BACE-1 and PSENEN. 

Small interfering RNAs (siRNAs) that can cause PTGS through RNA interference pathway have been designed. RNA analysis has been done to verify complementarity of antisense siRNA sequence with target mRNA sequence. Interaction studies have been done computationally between these antisense siRNA strands and seven Argonaute proteins. From the interaction studies, only one of the seven Argonaute proteins; 1Q8K, was found to have interaction with the siRNAs indicating the importance and uniqueness of this particular protein in RISC (RNA induced silencing complex). 

The interaction studies have been carried out for the microRNAs also. Out of the 700 mature human microRNAs collected, 394 microRNAs have been identified to show partial complementarity with their target sequence on PSEN-1 mRNA. Of these 394, five microRNAs have shown partial complementarity to early onset Alzheimer’s disease specific mutations in PSEN-1 mRNA. Interaction studies have been done between these microRNAs and Argonaute proteins. Thus, design, characterization and analysis of ncRNAs that contribute to post transcriptional gene silencing of Alzheimer’s disease have been achieved.
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Beyond BLASTing : ribonucleoprotein evolution via structural prediction and ancestral sequence reconstruction

Primary homology in DNA and protein sequence has long been used to infer a relationship between similar sequences. However gene sequence, and thus protein sequence, can change over time. In evolutionary biology that time can be millions of years and related sequences may become unrecognisable via primary homology. This is demonstrated most effectively in chapter 4a (figure 10). Conversely the number of possible folds that proteins can adopt is limited by the attractions between residues and therefore the number of possible folds is not infinite. This means that folds may arise via convergence between evolutionarily unrelated DNA sequences. This thesis aims to look at a process to will wring more information from the primary protein sequence than is usually used and finds other factors that can support or refute the placement of a protein sequence within the family in question. Two quite different proteins; the Major Vault Protein whose monomers make up the enigmatic vault particle and the argonaute family of proteins (AGO and PIWI) that appear to have a major hand in quelling parasitic nucleic acid and control of endogenous gene expression, are used to demonstrate the flexibility of the workflow. Principally the method relies on prediction of three-dimensional structure. This requires at least a partially solved crystal structure but once one exists this method should be suitable for any protein. Whole genome sequencing is now a routine practice but annotation of the resultant sequence lags behind for lack of skilled personnel. Automated pipeline data does a good job in annotating close homologs but more effort is needed for correct annotation of the exponentially growing data bank of uncharacterised (and wrongly characterised) proteins. Lastly, in deference to budding biologists the world over, I have tried to find free stable software that can be used on an ordinary personal computer and by a researcher with minimal computer literacy to help with this task

ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis.

Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5'-terminal nucleotide is important for sRNA sorting into different AGOs. Here we show that microRNA (miRNA) duplex structure also contributes to miRNA sorting. Base pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favours miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modelling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex-or miRNA-induced silencing complex (miRISC)-can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression. © 2011 European Molecular Biology Organization

Transfer RNA-derived small RNAs in the cancer transcriptome

The cellular lifetime includes stages such as differentiation, proliferation, division, senescence and apoptosis.These stages are driven by a strictly ordered process of transcription dynamics. Molecular disruption to RNA polymerase assembly, chromatin remodelling and transcription factor binding through to RNA editing, splicing, post-transcriptional regulation and ribosome scanning can result in significant costs arising from genome instability. Cancer development is one example of when such disruption takes place. RNA silencing is a term used to describe the effects of post-transcriptional gene silencing mediated by a diverse set of small RNA molecules. Small RNAs are crucial for regulating gene expression and microguarding genome integrity.RNA silencing studies predominantly focus on small RNAs such as microRNAs, short-interfering RNAs and piwi-interacting RNAs. We describe an emerging renewal of inter-est in a‘larger’small RNA, the transfer RNA (tRNA).Precisely generated tRNA-derived small RNAs, named tRNA halves (tiRNAs) and tRNA fragments (tRFs), have been reported to be abundant with dysregulation associated with cancer. Transfection of tiRNAs inhibits protein translation by displacing eukaryotic initiation factors from messenger RNA (mRNA) and inaugurating stress granule formation.Knockdown of an overexpressed tRF inhibits cancer cell proliferation. Recovery of lacking tRFs prevents cancer metastasis. The dual oncogenic and tumour-suppressive role is typical of functional small RNAs. We review recent reports on tiRNA and tRF discovery and biogenesis, identification and analysis from next-generation sequencing data and a mechanistic animal study to demonstrate their physiological role in cancer biology. We propose tRNA-derived small RNA-mediated RNA silencing is an innate defence mechanism to prevent oncogenic translation. We expect that cancer cells are percipient to their ablated control of transcription and attempt to prevent loss of genome control through RNA silencing

Nucleotide bias of DCL and AGO in plant anti-virus gene silencing

Plant Dicer-like (DCL) and Argonaute (AGO) are the key enzymes involved in anti-virus post-transcriptional gene silencing (AV-PTGS). Here we show that AV-PTGS exhibited nucleotide preference by calculating a relative AV-PTGS efficiency on processing viral RNA substrates. In comparison with genome sequences of dicot-infecting Turnip mosaic virus (TuMV) and monocot-infecting Cocksfoot streak virus (CSV), viral-derived small interfering RNAs (vsiRNAs) displayed positive correlations between AV-PTGS efficiency and G+C content (GC%). Further investigations on nucleotide contents revealed that the vsiRNA populations had G-biases. This finding was further supported by our analyses of previously reported vsiRNA populations in diverse plant-virus associations, and AGO associated Arabidopsis endogenous siRNA populations, indicating that plant AGOs operated with G-preference. We further propose a hypothesis that AV-PTGS imposes selection pressure(s) on the evolution of plant viruses. This hypothesis was supported when potyvirus genomes were analysed for evidence of GC elimination, suggesting that plant virus evolution to have low GC% genomes would have a unique function, which is to reduce the host AV-PTGS attack during infections

Molecular medicine of microRNAs: structure, function and implications for diabetes

MicroRNAs (miRNAs) are a family of endogenous small noncoding RNA molecules, of 19–28 nucleotides in length. In humans, up to 3% of all genes are estimated to encode these evolutionarily conserved sequences. miRNAs are thought to control expression of thousands of target mRNAs. Mammalian miRNAs generally negatively regulate gene expression by repressing translation, possibly through effects on mRNA stability and compartmentalisation, and/or the translation process itself. An extensive range of in silico and experimental techniques have been applied to our understanding of the occurrence and functional relevance of such sequences, and antisense technologies have been successfully used to control miRNA expression in vitro and in vivo. Interestingly, miRNAs have been identified in both normal and pathological conditions, including differentiation and development, metabolism, proliferation, cell death, viral infection and cancer. Of specific relevance and excitement to the area of diabetes research, miRNA regulation has been implicated in insulin secretion from pancreatic β-cells, diabetic heart conditions and nephropathy. Further analyses of miRNAs in vitro and in vivo will, undoubtedly, enable us determine their potential to be exploited as therapeutic targets in diabetes

The \u3cem\u3emir-51\u3c/em\u3e Family of MicroRNAs Functions in Diverse Regulatory Pathways in \u3cem\u3eCaenorhbditis elegans\u3c/em\u3e

The mir-51 family of microRNAs (miRNAs) in C. elegans are part of the deeply conserved miR-99/100 family. While loss of all six family members (mir-51-56) in C. elegans results in embryonic lethality, loss of individual mir-51 family members results in a suppression of retarded developmental timing defects associated with the loss of alg-1. The mechanism of this suppression of developmental timing defects is unknown. To address this, we characterized the function of the mir-51 family in the developmental timing pathway. We performed genetic analysis and determined that mir-51 family members regulate the developmental timing pathway in the L2 stage upstream of hbl-1. Loss of the mir-51 family member, mir-52, suppressed retarded developmental timing defects associated with the loss of let-7 family members and lin-46. Enhancement of precocious defects was observed for mutations in lin-14, hbl-1, and mir-48(ve33), but not later acting developmental timing genes. Interestingly, mir-51 family members showed genetic interactions with additional miRNA-regulated pathways, which are regulated by the let-7 and mir-35 family miRNAs, lsy-6, miR-240/786, and miR-1. Loss of mir-52 likely does not suppress miRNA-regulated pathways through an increase in miRNA biogenesis or miRNA activity. We found no increase in the levels of four mature miRNAs, let-7, miR-58, miR-62 or miR-244, in mir-52 or mir-52/53/54/55/56 mutant worms. In addition, we observed no increase in the activity of ectopic lsy-6 in the repression of a downstream target in uterine cells in worms that lack mir-52. We propose that the mir-51 family functions broadly through the regulation of multiple targets, which have not yet been identified, in diverse regulatory pathways in C. elegans

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells.

Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5\u27-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5\u27-tRNA halves as well, suggesting a previously uncharacterized link between 5\u27-tRNA halves and td-piRNAs. An increase in levels of the 5\u27-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5\u27-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to -1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs