Non-coding RNAs

What is a noncoding RNA?

A noncoding RNA is a functional RNA molecule that is transcribed from DNA but is not translated into a protein

What types of ncRNAs are short?

tRNA, microRNA, siRNA, piRNA, etc.

What kind of ncRNAs are long?

rRNA and more

microRNA General Notes

Short non-coding RNAs that repress gene expression post transcriptionally

What is the first step of miRNA biogenesis

Primary miRNAs

Transcribed here and capped and polyadenylated

Also cleaved by microprocessor (Drosha-Dgcr8) to create a hairpin

What is the second step of miRNA biogenesis?

Pre-miRNAs

It is now in the cytoplasm! Dicer is also invovled here

What is the third step of miRNA processing?

It forms a duplex (miRNA/miRNA*)

One strand goes onto RISC, and silencing happens with Argonaute

What are all the proteins involved in miRNA biogenesis?

RNA Pol II, microprocessor (drosha and Dgcr8), dicer, exportin 5, argonaute

What proteins are RNase II enzymes?

Drosha and Dicer

What proteins are dsRNA binding proteins?

Dgcr8

What happens if an miRNA is part of an exon?

It will likely affect the mRNA that it is orginiating from

What happens if an miRNA is part of an intron?

It will likely not affect the mRNA it originates from, as that final mRNA will liekly not have that sequence

What are the 4 ways that miRNAs regulate target genes?

Endonucleolytic clevage

deadenylation and degredation

Inhibition of translation initiation

Inhibition after translation initiation

What kind of miRNA regulation do mammals primarily use?

Endonucleolytic clevage

Deadenylation and degreadation

How do miRNAs target and recognize?

Perfect base pairing at seed region,

Bulge or mismatches in the central area

Good base paring between nucleotides 13 to 16 of miRNA

CLIP-seq

This sure is a process. I think its using a marked nucleotide to see where these miRNAs are binding by having them bind, degreading protein and seeing what was bound to that protein?

siRNA

Small interfering RNA are a class of 20-25 nucleotide long double stranded RNA molecules involved in the RNAi pathway

siRNA biogenesis

A LOT of their processing (if not most) happens in cytoplasm

It is dicer dependent and microprocessor-independent

How do endogenous siRNAs form?

Through transposable elements, cis ways (the 2 strands overlap in the DNA and are able to bind beacuse theyre really close to each other), trans (two unrelated ones have similar sequences), and hpRNA

How do exogenous siRNA and shRNA form?

1. Long dsRNA

2. Synthetic siRNA

3. Diced siRNA

4. Plasmid-based shRNA vector

5. Virus based shRNA vector

Target recognition

This is an odd slide. I would review it

Long non-coding RNAs (IncRNAs)

long (>200 nt)

They do not have an obvious ORF?

What are some features of IncRNAs?

Transcribed by RNA poly II

5’ capped and 3’ polyadenylated

Has exons and introns

What are the potential functions of IncRNAs?

3 of them:

Transcriptional noise

The transcription activity generates chromatin structure favorable for transcription of other genes

IncRNA molecules are functional

What do cis acting IncRNAs do?

They associate with the site of transcription and affect expression of neighboring genes directly!

They also affect the expression of neighboring genes

What do trans-acting IncRNA do?

They associate with DNA binding protiens or other regulator proteins

Localize and affect expression of target genes

Overall they work indirectly by promoting protiens to do specific things

How are IncRNA degraded?

They are decapped and deadenylated and lead to their degredation

NMD pathway is also involved here when they are in the cytoplasm

How are lncRNAs processed?

Capping, splicing, polyadenylation. Some lncRNAs undergo special processing events that are not seen in mRNA