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Lecture 3 GCATCCATCTTGGGGCGTCCCAATTGCTGAGTAACAAATGAGACGC TGTGGCCAAACTCAGTCATAACTAATGACATTTCTAGACAAAGTGAC TTCAGATTTTCAAAGCGTACCCTGTTTACATCATTTTGCCAATTTCG CGTACTGCAACCGGCGGGCCACGCCCCCGTGAAAAGAAGGTTGTT TTCTCCACATTTCGGGGTTCTGGACGTTTCCCGGCTGCGGGGCGG GGGGAGTCTCCGGCGCACGCGGCCCCTTGGCCCCGCCCCCAGTC ATTCCCGGCCACTCGCGACCCGAGGCTGCCGCAGGGGGCGGGCT GAGCGCGTGCGAGGCGATTGGTTTGGGGCCAGAGTGGGCGAGGC GCGGAGGTCTGGCCTATAAAGTAGTCGCGGAGACGGGGTGCTGGT TTGCGTCGTAGTCTCCTGCAGCGTCTGGGGTTTCCGTTGCAGTCCT CGGAACCAGGACCTCGGCGTGGCCTAGCGAGTTATGGCGACGAAG GCCGTGTGCGTGCTGAAGGGCGACGGCCCAGTGCAGGGCATCAT CAATTTCGAGCAGAAGGCAAGGGCTGGGACGGAGGCTTGTTTGCG AGGCCGCTCCCACCCGCTCGTCCCCCCGCGCACCTTTGCTAGGAG CGGGTCGCCCGCCAGGCCTCGGGGCCGCCCTGGTCCAGCGCCCG GTCCCGGCCCGTGCCGCCCGGTCGGTGCCTTCGCCCCCAGCGGT GCGGTGCCCAAGTGCTGAGTCACCGGGCGGGCCCGGGCGCGGG GCGTGGGACCGAGGCCGCCGCGGGGCTGGGCCTGCGCGTGGCG GGAGCGCGGGGAGGGATTGCCGCGGGCCGGGGAGGGGCGGGGG CGGGCGTGCTGCCCTCTGTGGTCCTTGGGCCGCCGCCGCGGGTC TGTCGTGGTGCCTGGAGCGGCTGTGCTCGTCCCTTGCTTGGCCGT GTTCTC Much of the genome remains to be annotated Non-coding • Non-protein coding Genes • cis-regulatory elements • Other functional sequences Protein Coding Human genome repeats The first non-coding RNA! The first non-coding RNA to be characterized was an alanine tRNA in baker’s yeast (Holley et al, Science, 1965). The “cloverleaf” secondary structure was revealed using X-ray crystallography in 1974. Robert Holly Ribosomal RNAs George Palade Abundant (80% of total RNA)! Humans : many copies of the rRNA genes in tandem repeats 300-400 rDNA repeats are localized on chromosomes 13,14,15,21 and 22 Euraryotes: Ribosomes 80S: 2 subunits Large (5S, 5.8S, 28S) 60S; Small (18S) 40S 28S, 18S and 5.8S rRNAs are made from a single transcript (45S) Venkatraman Ramakrishnan, Thomas A. Steitz, Ada E. Yonath http://www.nobelprize.org snoRNAs (small nucleolar RNAs) 150 species, 70-250 nt in length *Ribosomal RNA processing **rRNA modification (2'-Oribose methylation, or pseudouridylation) The majority of vertebrate snoRNA genes are encoded in the introns of proteins involved in ribosome synthesis or translation, and are synthesized by RNA polymerase II Small nuclear RNAs assembly U RNAs: U1, U2, U4, U5, U6 small nuclear RNA (for splicing mRNAs) first transesterification second transesterification Lariat intron released, debranched, degraded Other snRNPs released Other ncRNAs Enhancer-associated RNA (eRNA) Long non-coding RNAs Have intron/exon structures like protein-coding genes, but lack protein-coding potential. First several were discovered in early 90s’ H19, only transcribed from the maternal allele, is linked to Beckwith-Wiedemann syndrome (BWS) and cancer Xist, involved in X inactivation Beckwith-Wiedemann syndrome Long non-coding RNAs Tiling microarray experiments led to the finding that a large fraction of the mammalian genome is transcribed (Bertone, Science 306, 2242–2246 (2004).18. Kapranov. Science 316, 1484– 1488 (2007).19. Rinn,Genes Dev. 17, 529–540 (2003).20. Kapranov, Science 296, 916–919 (2002).) Bertone, Science 2004 Long non-coding RNAs Large scale full length cDNA cloning and sequencing efforts, such as the RIKEN’s FANTOM project, led to the finding that 62% of the mouse genome is transcribed, and the identification of thousands of long non-coding RNAs in early 2000s (Okazaki et al., Nature 2002, 420 (6915) pp. 563-73; Carninci et al., Science 2005, 309 (5740) pp. 1559-63). Non-coding RNA genes exhibit similar levels of conservation as the protein-coding genes at promoters. Many lncRNAs are dynamically expressed Carninci et al., Science 2005 Long non-coding RNAs identified by chromatin signatures H3K4m3 is a mark for promoters H3K36me3 is a mark for gene body of actively transcribed genes Genomic regions with H3K4me3 and H3K36me3 are likely genes Chromatin profiles can be obtained using ChIP-seq Long non-coding RNAs identified by chromatin signatures H3K4m3 is a mark for promoters H3K36me3 is a mark for gene body of actively transcribed genes Genomic regions with H3K4me3 and H3K36me3 are likely genes Guttman et al., Nature 2009, 458 (7235) pp. 223-7 Long non-coding RNAs identified by chromatin signatures LncRNAs have low protein coding potential LncRNAs are evolutionarily conserved LncRNA promoters are evolutionarily conserved Guttman et al., Nature 2009, 458 (7235) pp. 223-7 Protein coding potential of lncRNA Guttman & Rinn, Nature 2012, 482 (7385) pp. 339-46 Long non-coding RNAs LncRNA gene structures can be reconstructed from RNA-seq data using Scripture (Guttman et al., Nature Bioltech. 2010) On average the reconstructed lncRNAs have 3.7 exons, compared to 7 for protein coding genes Guttman et al. Nature Bioltech. 2010 vol. 28 (5) pp. 503-10 Long non-coding RNAs Cabili et al., Genes & Dev. 2011, 25 (18) pp. 1915-27 Properties of lncRNAs Tissue specific expression Lower abundance Functions of lncRNAs Guttman & Rinn, Nature 2012, 482 (7385) pp. 339-46 Many LncRNAs affect transcription either in cis- or. trans Xist HOTAIR Guttman & Rinn, Nature 2012, 482 (7385) pp. 339-46 Finding chromatin binding sites of lncRNA 48 complementary DNA oligonucleotides that were 20-mer each and tiled the entire length of HOTAIR ‘‘split-probe’’ strategy helps remove off target sequences. Glutaraldehyde crosslinking Chu et al., Molecular Cell 44, 667–678, 2011 Mode of action of lncRNAs CCND1 ncRNA GAS5 Guttman & Rinn, Nature 2012, 482 (7385) pp. 339-46 Proposed models of lncRNA functions Proposed models of lncRNA functions – modular principles Promoter associated small RNAs Enhancer associated RNA (eRNA) Bi-directional Short (few hundred bp to 1000bp) Lacks 5’ cap and 3’ polyA Transient and levels correlate with enhancer activities Functions unknown Kim et al., Nature 465,182–187(2010). Summary A large number long ncRNAs have now been found in the mammalian genome However, their functions are generally unknown, with the exception of a few. It is proposed that lncRNAs could function in a modular fashion to affect gene regulation, and other cellular processes. Clearly, one of the most exciting development in the future is to characterize their function. Small RNAs are regulatory!! ~20-30 nt long RNAs First small RNA, lin-4 microRNA (genetic screens in nematode worms) The number of small RNAs have exploded, and are implicated from heterochromatin formation to mRNA destabilization and translational control Diversity of small RNAs • siRNA Cellular gene silencing Generated by RNAi pathway 21-23nt Cleave target mRNA Target RNA from virus and transposons Generated by miRNA pathway 21-23nt •miRNA Cleave target mRNA inhibit translation initiation Differentiated cells NPC glia Function in many processes and cells neuron IGF/serum miR-34a •piRNA Dicer-independent 24-30 nt Fly, fish and mouse ovary and testis Derived from transposons and other repeated sequence elements ovary Self-renewing division MicroRNAs ~21-22 nt long non-coding RNAs More than 60% of protein-coding genes are predicted to be controlled by microRNAs Almost every cellular process investigated to date have a microRNA component microRNAs function in the form of ribonucleoprotein complexes (miRISCs: miRNA-induced silencing complexes). Ago, GW182 are components of miRISC Biogenesis and function of microRNAs ~160 miRNAs ~150 miRNAs ~1000 miRNAs Potential functions of long non-coding RNAs