Lecture 22 - Regulatory non-coding RNAs

Question 1

What type of RNA are non coding RNAs?

Answer 1

Transcripts that do not code for protein

• housekeeping ncRNAs (rRNA, tRNA, snRNA)
• regulatory ncRNAs (control gene expression)
• ncRNAs of unknown function

Question 2

What are the two types of regulatory non-codingRNAs?

Answer 2

Long non-coding RNAs (100s of kb)

Short non-coding RNAs

Question 3

Give an example of a long non-coding RNAs?

Answer 3

e. g. Xist ncRNA involved in dosage compensation (X-inactivation) in mammals
- switch of one X early in expression
- produced from the activated X chromosome
- often act as scaffolds to recruit epigenetic modifiers (histone modifications and DNA methylation) to the inactive chromosome

Question 4

What are the features of small ncRNAs?

Answer 4

• act as sequence specificity guides for effector complexes
• can influence gene expression at the transcriptional and post-transcriptional level (RNA level)
• MicroRNAs are a major class
• 1000s of sRNAs in higher eukaryotes
• produced by the RNA silencing/RNA interference pathway

Question 5

What is the process by which sRNAs are produced in the RNA silencing/RNA interference pathway?

Answer 5

• dsRNA is processed into sRNAs (21-25nts) by the DICER class of enzymes (RNAse III class)
• forms small dsRNAs with overhangs
• these then form into effector complexes, RNA-Inducde Silencing Complex (RISC), which is a nucleprotein, including:
• small RNA component (sequence specificty determinant)
• effector component (Argonaute class of proteins, endonuclease activity)
• Accessory proteins

Question 6

Who discovered RNA interference? And how?

Answer 6

Fire and Mello

• looked at gene expression in c.elegans
• Made GPT transgenic c.elegans
• injected with GFP dsRNA
• no fluorescence

Question 7

What is the trigger for targeted RNA degradation?

Answer 7

Double-stranded RNA triggers targeted RNA degradation of complementary transcripts
=RNA interferance

Question 8

What is RNA interference?

Answer 8

Mechanism that allows switching off of any gene through interaction with complementary sRNAs
-triggered by double stranded RNAs

Question 9

How do sRNAs result in surpression of gene expression?

Answer 9

small RNAs can direct mRNA cleavage or translational inhibition dependeing on the degree of complimentarity with its target (if high or almost exactly complimentary)

• if near perfect complimentarity with a coding region or UTR -> mRNA cleavage
• if have short complementary segment in 3’-UTR -> mRNA tranlational attenuation

Question 10

How can we identify the biological role of RNA silencing?

Answer 10

• how well conserved
• identify the phenotypic mutnats
• which regions of the genome produce small RNAs
• natural sources of sRNAs
• sequences of the small RNAs and what do they target

Question 11

What are the mutant phenotypes of RNAi in Arabiposis thaliana?

Answer 11

argonaute1 mutants
-effector complex mutated
-small, stunted growth, fail to develop properly
argonaute2 mutants
-hyper-susceptible to virus infection 

Therefore role of RNAi:

• control of endogenous gene expression
• defense against invasive nucleic acids

Question 12

What are the biological role of RNAi?

Answer 12

• control of endogenous gene expression

- defense against invasive nucleic acids

Question 13

What are the sources of dsRNA?

Answer 13

• read through transcription of inverted repeats e.g. from transposon elements, result in a fold back to form a ds structure
• replicating viruses
• via the copying action of RNA-dependent RNA polymerases (RDRs) (copy RNA into dsRNA)
• ‘Structured loci’ transcrirpts that have the potential to form secondary structures (e.g. miRNA)

Question 14

What two categories have cloning and sequencing of sRNAs of 21-24nts identifide?

Answer 14

• small interfering RNAs (derived from perfect dsRNA e.g. transposons, viruses, copying action)
• miRNAs (derived from imperfect hairpin structures)

Question 15

Where can animal miRNAs be derived from?

Answer 15

• mainly from RNA pol III transcripts
• a range of transcript forms that share the imperfect bulge hairpin structure
  e. g. miR-21 (1 hairpin)

Question 16

What is the process of miRNA biogenesis in animals?

Answer 16

• transcription of primary miRNA transcripts (pri-mRNAs) by RNA pol II (have long 5’ and 3’ ends)
• processing of the pri-miRNA to pre-miRNA by the Drosh enzymes
• export of the pre-miRNA into the cytoplams by the specific transporter complex (Exportin5)
• processing of the pre-miRNA by dicer to form the miRNA duplex
• unwinding of the duplex and loading into the RISC effector complex
• target mRNA cleavage or inhibition of translation

Question 17

Give an example of how miRNAs control developmental transitions?

Answer 17

• miR156 targets SPL3 mRNA
• SPL3 is a transcription factor that promotes the juvenile to adult transition in arabidopsis (no flowers -> flowers)
• miR156 supresses SPL3, consequentially supressing the juvenile to adult transition

Tested by increasing/decreasing miR156 activity

• over expression of miR156 by conjugating the sRNA to a strong promoter e.g. 35s promoter results in a delay in flowering
• expression of miR156 resistant form of SPL3 (by changing the nucleotide sequence by keeping the aa sequence the same) rsuls in an early flowering and a short juvenile phase
• expression of an mRNA that sequesters miR156 (dilutes the effect) results in early flowering and a short juvenile phase

Question 18

How are miRNAs involved in human disease?

Answer 18

• high percentage of human genes are targets of miRNAs
• any mis-regulation of miRNA expression can have far reaching consequences
• mis-regulation of miRNAs has been associated with many diseases (and proposed as biomarkers)
  e. g. Lin28 and let-7, miR200, miRNAs involved in cardiac disase

Question 19

Outline the involvement of Lin28 and let-7 miRNAs in cancer

Answer 19

• let-7 is an evolutionarily conserved class of miRNAs, negatively regulated by the RNA binding protein Lin28
• Lin28 is a pluripotency factor
• let-7 has important targets including oncogenes
• Lin28 levels are normally low in developing cells, leading to high Let-7 levels, allowing the supression of onco genes
• Lin28 level are elevated in cancer, supressing Let-7, leading to less supression of oncogenes (c-MYC, K-Ras and cyclin D1)

Question 20

How is miR200 involved in cancer?

Answer 20

• cancer progression has similarities with the epithelial-to-mesenchymal transition found during embryonic development in which E-cadherin (CDH1) is downregulated
• miR200 usually down regulates ZEB transcription repressors (which repress the expression of CDH1)
• in cancers expression of miR200 is often found to be switched off by DNA methylation
• reduction of miR200 rsults in increased levels of ZEB1 and ZEB2 and consequently reduced levels of CDH1 - increased cancer mestasis

Question 21

How are miRNAs involved in cardiac disease?

Answer 21

• cardiac myocytes respond to stress by undergoing hypertrophy
• miR133 is downregulated in diseased tissue
• miR133 normally supresses genes that promote hypertrophy
• hypertrophy like symptoms can be induced by supressing miR133

Question 22

What technique can be used to loook at miRNA levels?

Answer 22

Northern blot