PBEC 306 Small RNA Biology & Epigenetics Lecture 1 2024 PDF

PBEC 306 SMALL RNA BIOLOGY & EPIGENETICS LECTURE I 2024  Course Contents  Unit 1: Chromatin Modeling and Remodeling -- Polycomb complexes, SWI/SNF1 complexes and other chromatin modifiers.  Unit 2: Interpretation of DNA Methyla...

PBEC 306 SMALL RNA BIOLOGY & EPIGENETICS LECTURE I 2024  Course Contents  Unit 1: Chromatin Modeling and Remodeling -- Polycomb complexes, SWI/SNF1 complexes and other chromatin modifiers.  Unit 2: Interpretation of DNA Methylation Marks by Cellular Machinery -- Study of methylated DNA binding proteins, their structure and function, methods of altering DNA methylation.  Unit 3: Study of Histone Modifications -- Histone modifications, modifying enzymes, histone deacetylase inhibitors.  Unit 4: Chromatin Modification and Development -- Effect on somatic embryogenesis, leaf development, photosynthesis, flowering and ageing.  Unit 5: Epigenetics and Environment -- Role in plant stresses, epigenetic memory.  Unit 6: Epigenetics in Human Systems -- Role in immune response, cancer and cardiovascular diseases.  Unit 7: Non-coding RNAs -- Types and occurrence of non-coding RNAs, small RNAs in different biological systems, diversity and evolution of small RNAs.  Unit 8: Identification and Characterization of Small RNAs -- Discovery, detection and validation of small RNAs, target prediction and validation, databases on small RNAs, an overview of bioinformatics tools in small RNA biology.  Unit 9: Small RNA Pathways - Biogenesis of different classes of small RNAs, components and their characteristic features.  Unit 10: Regulation of Gene Expression by Small RNAs -- Transcriptional gene silencing (TGS), Post-transcriptional Gene Silencing (PTGS), gene activation, evolutionary transition of small RNA-target gene pair.  Unit 11: Biological Processes Regulated by Small RNAs -- Diverse roles of small RNAs in regulating biological processes in different organisms: bacteria, plants & animals, trans-kingdom cross-talk mediated by small RNAs.  Unit 12: Small Non-coding RNAs as Effective Tools in Biotechnology -- amiR technology, siRNA technology, Virus-induced gene silencing (VIGS), RNA Interference (RNAi) and RNA activation (RNAa), target mimicry, Short tandem target mimic technology (STTM) & miR sponges, CRISPR-Cas mediated genome editing technology, crop improvement, diagnostics and therapeutic applications in human diseases.  Unit 13: Hands-on Training -- Techniques for studying differential methylation of DNA, gene expression in response to altered DNA methylation, expression profiling of small RNAs, survey of small RNA databases, case studies of plant miRNA families. Central Dogma of Molecular Biology Storehouse Transmitter Workhorse Central Dogma: Original Version By Crick An Unpublished Note by Francis Crick in 1956 Molecular Cell. 2023; 83(3):315 It’s RNA World, After all ! Storehouse Transmitter Catalytic activity Gene Regulators Messenger Enzyme Activity Regulators Workhorses  Outline of Today’s Lecture 1. Introduction to non-coding RNAs 2. Housekeeping non-coding RNAs 3. Regulatory non-coding RNAs Small nc RNAs  Non-coding RNAs A ncRNA is the functional RNA molecule that is transcribed from DNA, but not translated into proteins. Different types of ncRNAs are known which are generally classified as:  Housekeeping Non-coding RNAs Housekeeping ncRNAs are abundantly and ubiquitously expressed RNAs that regulate generic cellular functions. They include rRNAs, tRNAs, snRNAs, snoRNAs, TERC. Usually small, ranging in size from 50 to 500 nucleotides long (except rRNAs). Generally constitutively expressed in all cell types and necessary for cell viability.  Ribosomal RNAs or rRNAs Most abundant class of RNAs in most cells, comprising approximately 80% of the cellular transcriptome. Ribosomes are remarkable ribonucleoprotein complexes that are responsible for protein synthesis in all forms of life. They polymerize polypeptide chains programmed by nucleotide sequences in messenger RNA in a mechanism mediated by transfer RNA. rRNAs serve as the essential binding site for ribosomal proteins within the assembled ribosome and contribute to the binding of extra-ribosomal factors and ribosome-associated proteins, resulting in the protein translation machinery. rRNAs were discovered during cell fractionation experiments. The subunits of ribosome and rRNAs were detected through differential centrifugation and that’s why they are identified by their rate of sedimentation (Svedberg coefficient).  Ribosomal RNAs or rRNAs Ribosomes are made up of large and small subunits and these two units come together for mRNA translation. Prokaryotes: rRNA of ~1500 nt in length with a Svedberg coefficient of 16S. Together with ribosomal proteins, the small subunit is of 30S. Large subunit has two rRNA molecules: one of approximately 2900 nt (23S) and the second one is shorter in length approximately 120 nt (5S). These two rRNAs together with proteins gives rise of large 50S subunit. Eukaryotes: Ribosomes are made of 60S and 40S subunits. Two short rRNA molecules less than 200 nt in length (5S and 5.8S) and two longer RNA molecules: one over 5 kb (28S) and another of nearly 2 kb (18S). Eukaryotic ribosome is of 80 S. In addition to this, eukaryotic cells have rRNA in mitochondria and chloroplasts.  Importance of rRNAs 1. Protein translation: The primary function of rRNA is in protein synthesis – in binding to messenger RNA and transfer RNA to ensure that the codon sequence of the mRNA is translated accurately into amino acid sequence in proteins and facilitate the process of translation. To achieve this, rRNA has a distinctive three-dimensional shape involving internal loops and helices that creates specific sites within the ribosome – the A, P and E sites. The P (peptidyl site) site is for binding a growing polypeptide, the A site (aminoacyl site) anchors an incoming tRNA charged with an amino acid (aminoacyl tRNA). After peptide bond formation, the tRNA binds briefly to the E site (exit site) before leaving the ribosome. In addition rRNA also has sites for binding to some ribosomal proteins and careful analysis has demarcated the exact residues in both the RNA and protein. 2. Evolutionary and diversity Studies: Ribosomal RNA are also expressed in every cell of all extant species. The sequence of the core catalytic sites are also highly conserved making rRNA an excellent tool for the study of taxonomy and phylogenetics. There is a difference in the rate of evolution of residues on the surface and interior of rRNA, and nucleotides involved in core catalytic activity, such as in the formation of a peptide bond, appear to have predated the appearance of life on earth. The extent to which two species differ in rRNA sequences can give a good estimate of their evolutionary distance. The 16S rRNA has become the molecular standard or taxonomic genomic marker in studying evolutionary relationships between almost all bacteria and archaea. The marker allows one to examine genetic diversity in microbial communities, specifically what microbes are present in a sample. However, the 23S rRNA has followed a very similar (if not identical) evolutionary path. The 23S rRNA therefore provides additional complementary data that can be tapped to study the evolution of the ribosome. The 16S and 23S rRNAs each have a high degree of sequence Identity with 30–40% of the well aligned positions between bacteria and archaea being conserved. Yet despite this large degree of identity, there are significant phylogenetic signals in the pattern of change of the remaining nucleotides that can reveal the evolutionary history of the molecules. 2-D structure of 16Sr rRNA. The length and position of stem- loops are very similar in different species, although the exact sequence may vary at several positions.  Ribosomal RNA sequences differ between species, due to mutation: The ribosome is an ancient and essential component of cellular organisms, and its form and function is consistent across the spectrum of living things. A key aspect of ribosomes and ribosomal RNAs is that their function is very highly “conserved”, or maintained by natural selection, between and among species. However, the molecules that make up the ribosome, including the ribosomal RNAs, differ subtly between species in their composition, due to differences (caused by mutation) in the sequences of the genes that encode them. Through variation in rRNA sequences we can distinguish organisms on approximately the species level and trace evolutionary relationships. However, the exact sequences of DNA that encode these components are not identical between organisms. Slight differences in the DNA sequence encoding these molecules can arise without altering their shapes significantly, and thus without affecting their function. The end result is that, over evolutionary time, organisms very slowly accumulate changes in the sequences of the genes that encode parts of the ribosome. 3. rRNA as Targets of antibiotics: rRNA is the most commonly exploited RNA target for small molecules. The bacterial ribosome comprises 30S and 50S RNP subunits, contains a number of binding sites for novel anti-bacterial agents. The large difference between prokaryotic and eukaryotic rRNA 16S rRNA enables rRNA-targeting against a broad spectrum of pathogenic bacteria. X-ray crystallography studies have elucidated many antibiotic-binding sites on the ribosomal subunit facilitating the design of novel antibiotics. It has also been shown that antibiotic resistance often stems from point mutations in these binding sites. For instance, the resistance of Euglena and E. coli to streptomycin stems from mutation in the 16S rRNA sequence. Similar results were found for the resistance of Streptomyces to Spectinomycin. 23S rRNA 4. Regulatory RNAs from rRNAs: In a new dimension to the function of rRNA, its precursors (pre-ribosomal RNA) have been implicated in the generation of micro RNA that mediate inflammation and cardiac disease in response to mechanical stress. The mechanisms of this activity are still being elucidated. For eg. miR-712 is generated from murine pre-ribosomal RN45S. This gene encodes for 45S which contains sequences for 18S, 5.8S and 28S rRNA with two intervening sequences. It is generated in DICER1-dependent manner and it induces endothelial inflammation and atherosclerosis (major underlying cause of cardiovascular pathologies and preferentially occurs in arterial regions) (Son et al., 2013; Nat Comm. 4, 3000).  Small Nuclear RNAs or snRNAs snRNAs are