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Functional Non-Coding DNA Part I Non-coding genes and non-coding elements of coding genes. BNFO 602/691 Biological Sequence Analysis Mark Reimers, VIPBG. What D oes ‘Functional N on-Coding DNA’ Mean?. DNA whose sequence affects transcripts made from DNA in some way

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functional non coding dna part i non coding genes and non coding elements of coding genes

Functional Non-Coding DNAPart INon-coding genes and non-coding elements of coding genes

BNFO 602/691

Biological Sequence Analysis

Mark Reimers, VIPBG

what d oes functional n on coding dna mean
What Does ‘Functional Non-Coding DNA’ Mean?
  • DNA whose sequence affects transcripts made from DNA in some way
  • Could affect transcription levels, splicing or sequestering of RNA
  • Three main ways to identify functional non-coding elements
    • Sequence characteristics – favored bases
    • Genomic conservation
    • Epigenetic marks and open chromatin
      • especially outside of genes
types of non c oding elements
Types of Non-Coding Elements
  • Non-coding RNAs
    • miRNAs, lncRNAs, etc
  • Non-coding gene elements
    • UTRs, splice sites, poly-adenylation sites, splice sites and regulating element, RNA-binding sites
  • DNA elements outside genes – our main focus
    • Promoters
    • Enhancers/Silencers
    • Insulators
types of non coding rna
Types of Non-Coding RNA
  • microRNAs
  • Silencing RNAs
  • Small nuclear/nucleolar RNAs
  • Piwi-Interacting RNAs
  • Long Non-Coding RNAs
  • Circular RNAs
  • Still other RNAs???
  • Comprehensive data base at www.ncrna.org
micro rnas
Micro-RNAs
  • Micro-RNAs are small non-coding RNA molecules, about 21–25 nucleotides in length
  • They are processed from much longer genes, or from introns within mRNA, by several molecular pathways
  • Micro-RNAs base-pair with complementary sequences within mRNA molecules, often in 3’ or 5’ UTR.
  • miRNA binding usually results in gene repression either via translational stalling or by triggering mRNA degradation

Image by Charles Mallery, U of Miami

micro rnas1
Micro-RNAs
  • The human genome encodes over 1500 miRNAs, which are believed to affect more than half of human genes
  • miRNAs are abundant in many cell types
    • Thousands of copies per cell of some miRNAs
    • Those within gene introns share regulation
  • miRNAs are well-conserved across vertebrates
    • No orthologs between plant and animal miRNAs
    • miRBase is the comprehensive repository of micro-RNAs
other short rnas sirna
Other Short RNAs: siRNA
  • Small interfering RNAs are double-stranded with an overhang
  • They are processed by some of the same machinery as miRNAsand have some of the same effects
other short rnas p irna
Other Short RNAs: piRNA
  • Piwi-Interacting RNAs are longer 26-31 base single-stranded RNAs
    • PIWI (P-element Induces Wimpy Testis) protein
  • Over 50,000 sequences known in mouse
    • They are the largest class of nc-RNA
  • They seem to play an ancient role in defenseagainst retro-viruses and transposons
other short rnas snrnas snornas
Other Short RNAs: snRNAs & snoRNAs
  • Small nuclear RNAs (snRNAs) are typically ~ 150 bases long, and associate with protein
    • Many conserved copies of each snRNA gene
    • U1-U6 snRNAs key parts of splicing machinery
  • Small nucleolar RNAs (snoRNAs)
    • Guide chemical modifications of other RNAs
    • Prader-Willi syndrome results from deletion of region containing 29 copies of SNORD116 on chr 15q11

U6 snRNA

long non coding rnas
Long Non-Coding RNAs
  • Many long (>200bp) stretches of genome are transcribed and have epigenetic marks like those of protein-coding genes
  • Most of these are spliced RNAs with two (or more) exons
  • GENCODE v15 has 13.5K lncRNA
  • See also
    • Derrien et al, Genome Research 2012
    • Lee, Science 2012

From Derrien et al Genome Res 2012

many lncrnas induce silencing
Many lncRNAs Induce Silencing
  • Coat nearby gene(s) and silence them
  • Xist binds to gene clusters first
  • Xist binds disparate parts of chromosome
  • Many lncRNA are antisense to genes
  • Some lncRNAs maintain pluripotency of stem cells

From Jeannie Lee lab (Harvard) website

long non coding rnas 2
Long Non-Coding RNAs - 2
  • Most lncRNAs are expressed in only a few tissues
  • Most human lncRNAs are specific to the primate lineage

From Derrien et al Genome Res 2012

circular rnas
Circular RNAs
  • Several thousand non-coding RNAs apparently form circular structures
  • Many form complexes with AGO and seem to absorb attached miRNAs, blocking processing
  • CDR1 has 70 conserved binding sites for mir7
functional pseudo genes
Functional Pseudo-Genes
  • Pseudo-genes are copies of genes that are decaying and rarely (never) make proteins
  • Some pseudo-genes act to absorb negative regulators of the original gene – eg. SRGAP2B
how to identify non coding rnas
How to Identify Non-Coding RNAs?
  • Short (and long) RNA transcriptomes
  • Promoter chromatin marks for independent (non-embedded) miRNAs and lncRNAs
non coding elements of genes
Non-Coding Elements of Genes
  • TSS
  • 5' UTRs
  • Introns
  • Splicing regulation sites
  • 3' UTRs
  • Termination/Poly-adenylation sites
transcription start sites
Transcription Start Sites
  • Transcription of most genes may initiate at several distinct clusters of locations with distinct promoters for each TSS
  • Two major types of metazoan TSS: CG-rich broad TSS, and narrow (often tissue-specific) TSS
transcription start sites1
Transcription Start Sites

Transcription often starts at CG within promoter

5 untranslated regions
5’ Untranslated Regions
  • First exon often contains dozens to thousands of bases before Start codon (median 150)
  • Sometimes contains regulatory sequences, e.g. binding sites for RNA binding proteins, and translation initiators
splice regulatory sites
Splice Regulatory Sites
  • Splicing is achieved through binding of spliceosome to recognition sequences on nascent RNA molecule
splice regulatory sites1
Splice Regulatory Sites
  • Tissue-specific splice regulatory sites are highly conserved

From Merkin et al Science 2012

non coding elements in coding exons
Non-Coding Elements in Coding Exons
  • Many regulatory sites occur within coding exons, esp. toward 5’ end
  • These constrain some codons as much as protein sequence
  • Many human SNPs break TFBS but have little effect on protein (AFAWK)

From Stergachis et al Science 2013

3 untranslated regions
3’ Untranslated Regions
  • Longest exon is usually 3’UTR (>1000 nt)
  • Typically 1/3 – 1/2 of a gene is in 5’ & 3’ UTRs
  • 3’UTR has binding sites for miRNAs and RNA binding proteins
  • AU-rich elements (AREs) stabilize mRNA
  • Proteins recognize complex secondary structure

GRIK4 3’UTR secondary structure is conserved

rna binding protein sites
RNA Binding-Protein Sites
  • mRNAs are usually further processed (e.g. transported or sequestered)
  • RNA binding proteins recognize specific motifs within secondary structure of 3’ or 5’ UTR
  • These sites are often highly conserved

From Ray et al Nature 2013

poly adenylation termination sites
Poly-adenylation/Termination Sites
  • Transcripts can be terminated and poly-adenylated at sites with specific sequences
  • Most genes have alternate poly-adenylation sites
  • Median lengths of 3’UTR are 250 & 1773 bp(mouse)
poly adenylation termination sites1
Poly-adenylation/Termination Sites
  • Rapidly proliferating cells express gene isoforms with short 3’ UTRs
  • Neurons typically have longer 3’ UTRs

Types of alternate poly-adenylation

Elkon et al, NRG 2013