Epigenetic and non-coding RNAs

What are CpG islands

surround housekeeping genes (need to be kept on all the time – important) that code for proteins that keep cells viable 

• Helps maintain cellular specialization 

• CpG methylation is important for efficient gene repression to be passed onto daughter cells 

• Surround the promoter 

  • 20,000 of CpGs mark the 5’ end of transcription units (typically around the promoter) 

• This ratio probably reflects a balance between methylated CG loss by DNA repair and CG gain by random mutation. The CG sequences that remain are very unevenly distributed in the genome; they are present at 10 times their average density in selected regions

Epigenetic inheritance

heritable changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence 

• Epi = in addition to => environmental 

• Can be affected by the environment 

  Ex: Lactose tolerance 

  • LPH metabolizes lactose (encoded by the LCT gene on chr. 2) 

    • LCT expression regulated by polymorphism in upstream gene MCM6 (if polymorphism is missing, then lactose intolerance happens) 

  • LPH is high in European populations and lower in African populations => likely because they need more vitamin D than Africans  

• cis and trans epigenetic mechanisms

What are cis-epigenetic mechanisms

within the same DNA strand/molecule 

1. DNA methylation 

2. Histone modification 

What are trans epigenetic mechanisms

something outside the sequence that’s affecting the gene 

1. Positive feedback loop activated 

2. Conformation change to aggregated state 

What is imprinting

= silencing 

• A small minority of genes are expressed if inherited from mother, some expressed if inherited from father 

• Imprinting genes = one gene is silenced by methylation and cannot be transcribed 

  • Normal inheritance = 2 working copies of a gene (one from each parent) 

• Imprinting DOESN’T cause a disease, the other remaining gene does 

• Disease: Prader-Willi & Angelman syndrome (imprinting of gene in chr. 15 & a deletion) 

What is RNA interference (RNAi)

Short single-stranded non-coding RNAs of 20-30 nucleotides that guide or interact with other RNAs in the cell 

• Can inhibit translation of mRNA by catalyzing its destruction 

• Can mess with RNA and template during transcription and form repressive chromatin on the DNA 

• Proteins involved in RNAi are also involved in breaking apart dsRNA to form 23 nucleotide long siRNA 

• 3 types (miRNA, siRNA, piwiRNA) 

What are non-coding RNAs

lncRNAs = act as scaffold molecules (hold proteins together – after splicing) 

• Longer than 200 nucleotides 

• Ex: Telomerase RNA, Xist RNA, silencing RNA, gene regulators

What is CRISPR

Viral DNA fragments are incorporated into bacterial genome, acting as a vaccine to make small noncoding RNAs (crRNA) 

• crRNAs destroy the virus if it reinfects the bacteria 

  • Now using miRNA/siRNA (these are the Cas proteins that will interact with crRNA) 

    • Cas proteins need a PAM sequence to recognize bacteria’s genome and cleave in the right place 

What does DNA methylation do

Methylation = methylating Cytosine 

• marks genes that are “imprinted” 

• Requires a methyl transferase to pass along the methylation to correct daughter cells 

• Regulate gene expression (without changing DNA sequence) 

• Methylation of CpG sequences is important in efficient gene repression that can be passed on to daughter cells 

  • Helps maintain cellular specialization 

• Occurs right after DNA replication (inherited) 

  • Methyl transferase recognizes methylated C at CpG and adds it to new daughter strand 

What is deamination

Deficient in mammals because the methylated cytosine mutate into a T over time (via accidental deamination) 

• The remainder, C, are present in CpG islands in selected regions of the genome 

What are the 3 classes of RNAi

miRNA, siRNA, piRNA

What is micro RNA (miRNA)

Regulate some of human protein-coding genes by forming base pairs (complementary bp usually 7bp long in 3’ UTR of mRNA) with certain mRNAs & fine tune translation 

• Shut down method 

  • Small but mighty 

• Regulate gene expression by repressing translation via mRNA degradation 

  • miRNA calls other gene regulators to further reduce translation (mRNA of interest MUST have short common sequence in their 3’ UTR) 

• Made by RNA pol. II with a cap and poly-A tail. Proteins (Argonaute) are then added to form RISC (RNA-induced silencing complex) 

  • RISC = proteins + miRNA complex 

• Involved in heterochromatin formation using methylated histones

What are small interfering RNA (siRNA)

Look for viral or transposable RNA element (transcriptional repressor) (defense mechanism?) 

• Self-replicating: RNA pols use siRNA as primer to make more precursor siRNAs 

  • Broken apart and destroyed by RITS 

• Considered to be an ancient form of RNA interference  

What are Piwi-interacting RNA (piRNA)

bind to piwi proteins and protect the germ line from transposable elements 

Explain viral protection in relation to CRISPR

bacteria use non-coding RNA to destroy the DNA of invading viruses (via CRISPR) 

• Viral DNA fragments are incorporated into bacterial genome, acting as a vaccine to make small noncoding RNAs (crRNA) 

  • crRNAs destroy the virus if it reinfects the bacteria 

    • Now using miRNA/siRNA (these are the Cas proteins that will interact with crRNA) 

      • Cas proteins need a PAM sequence to recognize bacteria’s genome and cleave in the right place 

  • CRISPR is now used in plants and animals to manipulate genomes

What is Prader-Willi syndrome

aused by deletions of genetic region that includes small nuclear ribonucleoprotein polypeptide N gene 

• Maternal copy of gene is silenced (imprinted) 

  • Paternal gene is deleted 

• Increased appetite (over-weight) 

What is Angelman syndrome

Caused by a loss of expression of a single maternally expressed gene in the same region: UBE3A 

• UBE3A encodes for E3 ubiquitin ligase protein (involved in targeting proteins for degradation & is only imprinted in the brain) 

• Loss of UBE3A = abnormalities in normal protein degradation during brain development 

• The paternal gene imprinted 

  • Maternal is loss 

• “smiling” all the time