L13: RNA processing Flashcards
1
Q
Types of RNA processing events
A
- Addition of a 5’ -cap
- Addition of a 3’-tail
- Splicing (usually)
- Editing (sometimes)
2
Q
RNA cleavage
A
- Processing rRNA and tRNA from precursor RNA molecules into correct length by ribonucleases
-> ensures they are available in the same amounts within the cell
3
Q
Functions of the 5’ cap
A
- Protection of mRNA from degradation
- Translatability (presence of cap stimulates translation of mRNA ~300-fold
- Transport from the nucleus to the cytoplasm
4
Q
Capping process (3 steps)
A
Following pausing…
1. An RNA triphosphatase removes the terminal phosphate at the 5’ end
2. Guanylyl transferase uses GTP to attach GMP
3. The guanine is methylated by a methyltransferase
5
Q
Polyadenylation
A
- First step in termination is synthesis past this site
- Up to 70 nts long; AAUAAA…CA…{U- or G- rich region}
- mRNA is cleaved after the CA
- The 3’ end then has adenosines added by poly(A)polymerase
6
Q
Splicing
A
- More complexity in organism generally means more spicing of introns (introns found far more frequently in euk than prok)
- Removed by transesterification reactions; (in metazoa) most are removed by spliceosome (composed of proteins and snRNAs), although some are self splicing
7
Q
Intron excision/ splice site recognition
A
- Intron is excised as a lariat structure following two consecutive transesterification reactions
- Must be a GU at 5’ end and a 3’ AG; defines intron boundary
- The boundaries are recognised by a spliceosome, made up of 5 small ribonucleoproteins (snRNPs), each of which contains a snRNA (100-300 nt) and proteins
8
Q
The EJC complex (function)
A
- Exon junction complex; left at splice junctions after splicing
- Marks the transcript as processed and interacts w/ export and translation proteins
-> prevents incompletely processed RNA from being exported
9
Q
Why is splicing valuable? Regulation of alternative splicing
A
- Alternative splicing produces variation; can produce different versions of the mRNA
-> 90% of human genes have alternative splice forms - Distinct mRNAs from same pre-mRNA
- Developmentally controlled splicing
- Organ/tissue specific splicing
10
Q
RNA pols and splicing
A
- RNA pol II coordinates capping, splicing and ploly-adenylation
11
Q
RNA editing (example organism w/ mechanisms)
A
- Process that adds/deletes bases from pre-mRNA or chemically alters bases
-> bases don’t match DNA seq.
e.g. trypanosomes… - Organisms found in bloodstream
- Their mitochondrial genomes are relatively highly edited
- Post-transcriptionally add and remove bases form mRNA
- Mechanism utilises gRNA (guide), either recruits endonuclease then uridylyltransferase (adds U’s to 3’ end) then ligates fragments
- Alternatively, gRNA recruits endonuclease then exonuclease to cut out bulged U in mRNA, then ligates
12
Q
Substitution editing in humans (example)
A
- Apolipoprotein-B (gene contains 29 exons, total 4564 codons. Codon 2153 is CAA, codes for aa glutamine)
- Transcribed in liver cells (4563 aas) and in intestinal cells (additional step of pre-mRNA processing occurs: the deamination of the C nt in Codon 2153 into a U -> stop codon)
-> apolipoprotein B-48, 2152 aas
13
Q
RNA degradation
A
- RNAs have varying stability; one determinant of stability in euk is the presence of the poly-A tail
-> removal is directed by AU-rich elements (AREs) in 3’ UTR (untranslated region) of some mRNAs - AREs are often found in the mRNAs that are only required for a very short period of time
14
Q
mRNA degradation by RNAi
A
- RNA interference, first identified in nematode worms
- Injected dsDNA into worms, found corresponding gene was down-regulated
- in euk, 2 types of sRNAs allow this…
- Short interfering RNAs
- MicroRNAs (derived from RNA pol II transcripts
15
Q
miRNA processing
A
- Initially produced from RNA pol II: long regions of self-complementality -> hairpin, basepairing within molecule
- Drosha complex and DGCR8 bind, cleaving cap and tail, producing precursor miRNA -> exported from nucleus
- Dicer and TRBP2 cleave hairpin end
-> miR:miR - Alternatively, exogenous or endogenous dsRNA is cut into siR
- The miRNA or siRNA are loaded onto argonaute (Ago), one strand is degraded, producing mature miRISC/siRISC
