Augustus Barbosa Posttranscriptional Control Flashcards
What are the three modification steps which occur in RNA modification in eukaryotes, and what do these steps ensure?
The addition of the 5’ methyl cap
The removal of introns
The addition of a Poly A. Tail
These modifications ensure that the RNA is always bound to a protein (collectively termed RNP) this prevents the formation of thermodynamically stable double stranded RNA which is inaccessible to cellular machinery
The proteins are RNP1, RNP2 which have a structurally conserved domain containing a recognition motif of exposed, positively charged beta sheets
What occurs with the addition of the 5’ cap?
This process is unregulated, occurs shortly after translation and must occur correctly for translation to occur
RNA triphosphatase cleaves the gamma phosphate
RNA guanylyltransferase adds a Guanine to the beta phosphate
RNA methyltransferase adds a 7 methyl cap to this
What occurs with RNA splicing?
5 Proteins involved
U1 and U2 associate to RNA via base pairing
U4, U6, U5 join resutling in spliceosome formation
U4, U6 catalyse first esterification
Spliceosome is removed
Co-transcriptional process where spliceosomes cross talk with RNA pol. II
Spliceosomes regulate the splicing process and create degeneracy which allows for alternative splicing patterns
Alternative splicing is where repressors bind to specific RNA sites preventing them from being spliced causing the formation of a different mRNA and consequently a different functional protein
This is mediated through non-cryptic introns (where there is a very conserved splicing signal) and cryptic intorons where there is a very poorly conserved signal sequence
What occurs with the addition of the poly A. tail?
Heterotetramer ‘cleavage and polyadenylation specific’ binds to AAUAAA
However this is unstable, and requires biding of a G/U rich motif for stabilization, cleavage factors bridge these two proteins to recruit the poly A tail
Poly A binding protein allows for regulation of the poly A. tail as does the alternative cleavage sites, the length of the added tails and cytoplasmic polyadenylation and deadenylation events
What is RNA editing?
A process which occurs in tRNAs where bases and genetic codes are changed, allowing the formation of a unique secondary structure of RNA
Adenosine deaminase and Cytosine deaminase are the two key enzymes in this process
How does nuclear transport play a role in gene expression?
As eukaryotes have their genetic material sequestered in the nucleus, the mRNAs must be transported out of the nucleus to undergo translation
This allows for additional surveillance as mRNAs travelling out of the nucleus can be monitored to prevent translation of potentially harmful proteins
How does nuclear transport occur?
The nuclear pore complex conatins a meshwork of FG nucleoporins
This prevents molecules greater than 40kDa from moving through the poor via osmosis
mRNAs require a transporter called mRNA exporter this molecule has an exterior structure which interacts with the FG meshwork to splitt them apart to allow movement across the pore
How is surveillance of materials moving across the nuclear pore achieved?
the mRNA is stamped as ready for transport through presence of Cap binding complex, PABPII, SR proteins and RNA export factor
These complexes are not found in misprocessed RNAs which prevents binding of the transporter and exposes the mRNA to enzymatic degradation
Viruses can take advantage of this system through use of a Rev-Response element motive to bind Rev and hijack the exporter protein
How is gene expression controlled in the cytoplasm?
RNA silencing is achieved through the use of miRNA or SiRNA which generates double stranded RNA which cannot be translated
How do miRNAs controll gene expression
miRNAs are present to regulate endogenous genes, they are produced by a long precursor which forms an imperfect hairpin loop, this results in cleavage of the mismatch forming premiRNP, this is then cut more specifically and targeted
miRNAs typically bind at the 3’ UTR with multiple targets, the inihbition of translation occurs via competiton for cap binding and/or translation initiation factors, blockage of circularisation, ribosomal drop off, promint degradation
How do siRNAs control gene expression?
Defense against invasive nucleic acids, these have a different source to miRNA, typically using an exogenous source reulsting in this being an effective laboratory tool as it can regulate gene knock out
Triggered by perfect double stranded RNA, base pairs at 5’ end of target mRNA, argonaute will then make a nick in the invasive RNA allowing it to be degraded via exoenzymes
What is cytoplasmic polyadenylation of mRNAs?
Some RNAs are stored in a translationally dormant state via a short tail and a constitutive repressor and the AAUAAA signal sequence
These results in recruitment of maskin preventing the action of PABP I initiating transcription
If progesterone phosphorylates the CPEB then maskin will dissociate from the mRNA resulting in extension of the mRNA and the initiation of translation
How are mRNAs degraded in the cytoplasm?
Occurs via deadenylation-dependant pathway, which is controlled via suicide signals seen in the mRNA which cause endolytic cleavage and exonucleic decay
How is protein synthesis globally regulated?
Occurs when a cell is under nutritional stress and must down regulate it is activity for energy conservation
This is achieved via the TOR (Target of rapamycin) pathway where the TOR protein uses phosphorylation to either activate or inhibit protein activity
When in the normal state TOR binds GTP, while in the starved state this is a GDP
How are rRNAs proccessed?
Most ribosomal subunit synthesis occurs in the nucleolus, transcription of rRNAs is multicistronic in eukaryotes
prerRNA is extensive, endo/exonucleases and nucleotide modfication all occur, guided by U3snoRNA, RNAseMRP, snoRNAs..)
Pseudouridylation and methylation are the most common modifications
