Types of ncRNA (4)
- tRNA: transfer RNA
- rRNA: ribosomal RNA
- snRNA: small nuclear RNA
- snoRNA: small nucleolar RNA
What is RNP and which cellular processes is it involved in?
Ribonucleoprotein complexes: cellular process which RNA is involved in like
- DNA replication
- RNA processing
- Protein synthesis
- Protein targeting
- Catalase chemical rxns in cells
- Gene regulation
The roles of snRNA
assist in pre-mRNA splicing: splice out introns using spliceosomes (U1, U2, U4, U5, U6). 1x snRNA + multiple proteins = snRNP
- 5’ splice site recognition
- Branch point site
- catalyse 5’ splice site cleavage by bringing 5’ site close to branch point
With the use of snRNA how is pre-mRNA modified?
- U1 attaches to 5’ splice site
- U2 attaches to branchpoint (Adenine)
- U4, U5, U6 binds at intron region to complete spliceosome assembly
- 5’ splice site is cut and attaches on A (branchpoint)
- U1 + U4 released & U5 (hold intron) + U6 shift positions
- 3’ splice site is cut & exons covalently bond to each other
The role of snoRNA
involved in rRNA processing process & chemical modification of rRNA
With the use of snoRNA how is rRNA modified?
- snoRNP binds to specific sites in pre-rRNA to modify bases (adding methyl & pseudouridine groups)
- Cleavage w/ nucleases into mature rRNA
How piRNA is involved in transposon silencing?
- Transcription of piRNA clusters => pre- piRNA (ss)
- Primary processing of pre- piRNA => shortened
- Protein binds on piRNA => PIWI proteins (AgO2 endonuclease)
What is RNA interference (RNAi)
mediated by siRNA (short interfering) & miRNA (micro interfering). Short dsRNA degrades mRNA (not able to be translated)
How did Jorgensen experiment suggest RNAi was occurring?
Trying to cultivate flowers w/ extra purple pigment, he injected additional pigment-producing gene = purple + white flower. (Addition of genes = triggered defence mechanism)
Explain Fire and Mello’s experiment on silencing the muscle protein gene on a roundworm
- Silencing worked when dsDNA was injected but not on ssRNA (sense + antisense) = unfunctional gene = muscle function loss = twitching
- dsRNA had to match mature mRNA sequence (on post transcription= meaning introns/promoter sequences triggered it)
- only a small amount was required to trigger is
Explain how siRNA triggers gene silencing
- long -> short (21-22nt) dsRNA w/ dicer (member of RNase III)
- 2x ssRNA (guide + passenger strands)
- guide strands incorporated in RISC
- post-transcript. gene slicing by Argonaute: guide strands 100% matches sequence in mRNA
Explain how miRNA triggers gene silencing
- pre-miRNA (ss, imperfectly base-paired hairpin)->19-23nt miRNA
- miRNA binds onto RISC
- ANIMALS: repress translation w/out mRNA cleavage by blocking ribosomes (PARTIAL complementary = targets multiple mRNA = many genes affected)
Differentiate between the RISC (RNA-induced splicing complex) assembly and the effector step.
- RISC: guide strand incorporated in RISC
- Effector: guide strands complementary bind to mRNA => repress translation by cleavage or block ribosome
Uses of RNAi to suppress genes.
Suppress genes = look for phenotypic effect in:
- Testing hypothesis of gene function
- Functional screening & target identification
- Target validitaion
- siRNA as therapeutics
Identify the difference of miRNA in plants and animals.
- ANIMALS: repress translation w/out mRNA cleavage by blocking ribosomes (PARTIAL complementary = targets multiple mRNA = many genes affected)
- PLANTS: (like siRNA) perfect complementary to binding sites => mRNA cleavage.
In which cells will siRNA cause an interferon response?
Mammalian cells: when dsRNA >30bp = nonspecific antiviral interferon response
Explain the differences between siRNA and miRNA triggering gene silencing: configuration of RNA & complementarity of guide strand
Configuration: mi= ssRNA, si= dsRNA
Complementarity: mi= not exact (1= target 1+ mRNA), si= 100% perfect match
How is siRNA/miRNA directed to RISC components?
Dicer combines w/ dsRNA proteins (TRBP + Ago2)
