L13: RNA processing Flashcards
Types of RNA processing events
- Addition of a 5’ -cap
- Addition of a 3’-tail
- Splicing (usually)
- Editing (sometimes)
RNA cleavage
- Processing rRNA and tRNA from precursor RNA molecules into correct length by ribonucleases
-> ensures they are available in the same amounts within the cell
Functions of the 5’ cap
- Protection of mRNA from degradation
- Translatability (presence of cap stimulates translation of mRNA ~300-fold
- Transport from the nucleus to the cytoplasm
Capping process (3 steps)
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
Polyadenylation
- 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
Splicing
- 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
Intron excision/ splice site recognition
- 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
The EJC complex (function)
- 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
Why is splicing valuable? Regulation of alternative splicing
- 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
RNA pols and splicing
- RNA pol II coordinates capping, splicing and ploly-adenylation
RNA editing (example organism w/ mechanisms)
- 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
Substitution editing in humans (example)
- 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
RNA degradation
- 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
mRNA degradation by RNAi
- 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
miRNA processing
- 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
Gene silencing by miRISCs vs siRISCs
miRISCs
- Typically bind to 3’UTR of mRNA, don’t directly cleave RNA (not totally homologous to target)
- Repress translation (mechanism not understood)
- Activate deadenylation, decapping and exonucleolytic degradation
siRISCs
- Make perfect base-pairs w/ mRNA; Ago cleaves target mRNA -> further degraded
-> selectable, targetable degradation of mRNA; important tool for study
