Long and small non-coding RNAs
10 important questions on Long and small non-coding RNAs
What are the roles of long and small non-coding RNAs?
• microRNAs: fine-tuners of gene expression
• siRNAs/piRNAs: genome stability –silencing repetitive DNA, protection against viruses
What is the effect of IncRNAs in transcription?
- Transcription in antisense direction interferes with transcription protein coding gene = transcriptional interference
Antisense IncRNAs can inhibit expression by ANRIL
- Antisense ANRIL lncRNA recruits Polycomb proteins (PRC1 and PRC2), resulting in silencing of the protein coding genes CDKN2A and CDKN2B
How does HOTAIR act?
- HOTAIR binds to both PRC2 (Pc protein complex; adds H3K27me to target loci) and to H3K4me2/3 demethylase (LSD1; removes H3K4me2/3)
- repress multiple target genes in genome
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What is the role of Xist and Tsix?
- Xist depends on PcG proteins to spread over Xi
Tsix prevent X-inactivation on active X
How can the function of IncRNAs be tested?
- ncRNA-a7 functions as scaffold for assembly transcription factor complexes
- ncRNA-a7 knockdown by small RNAs leads to decreased expression of target gene
- HOTTIP guides WDR5/MLL complex (H3K4 methyltransferase) to transcription start sites
- HOTTIP knockdown by small RNAs leads to decreased expression HOXA target genes and loss H3K4me3 across the locus
What are the role of small RNA?
- double-stranded RNA (ds-RNA) is the primary trigger of RNAi
- Multiple transgenes inserted in inverted repeat (IR) orientation trigger silencing
- IR transgenes produce dsRNA
Which proteins are involved in RNAi?
- Dicer(-like) (DCL) proteins cleave long dsRNA molecules into small dsRNA fragments
RISC
- The small dsRNAs associate with ARGONAUTE (AGO) proteins in a RISC complex
- The dsRNAs are unwound & one strand remains bound to AGO
- The activated RISC complex binds target RNA, and cleaves target RNA/blocks translation/results in heterochromatin
What are the different outcomes of the small RNA pathways?
- Post-transcriptional gene silencing (PTGS)
- RNA is degraded or not translated
- transcription of the gene is unaffected
- miRNA. PiRNA, siRNA
- by affecting mRNA translation or stability
- Transcriptional gene silencing (TGS)
- No RNA is made
- piRNA, siRNA
- hrough epigeneticfont-size modification of chromatin
What is the role of piRNA in TGS?
•piRNAs mediate DNA & H3K9 methylation of TEs in the nucleus
•piRNAs result in decay TE RNA and transcriptional silencing TEs
•piRNAs are mainly present in germ line cells
•piRNAs mediate genome stability in germline flies, zebrafish & mice by silencing TEs & other repetitive DNA
What is the role of piRNA in PTGS?
silencing TE-derived RNA (PTGS)
piRNA are mainly encoded by ‘piRNA clusters’ (contain TE sequences)
•Single-stranded RNA precursors are processed into piRNAs (24-35 bp)
•piRNAs bind to TE RNAs & complex with Argonaute-type proteins (PIWI, AUB(ergine), AGO3)
•Protein complex cleaves TE RNAs in cytoplasm
•Primary piRNAs are amplified in ‘ping- pong’ amplification cycle in cytoplasm
• primary piRNAs bind to TE RNAs and lead to new, secondary piRNAs, etc
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