Eukaryotic Gene Regulation Flashcards

Question

action of alternative splicing regulators?

Answer 1

regulation in cell specific way: Cell 1 - does not express repressor even though it has repressor site next to splice site so splicing goes ahead ther Cell 2 - expresses repressor so does bind repressor site blocking splicing machinery same but other way around for activator presence of given activator/repressor key for splicing activity

Answer 2

alternative splicing does not only affect the OFR alternative last exon - UTR sequence comes after last exon an alternative last exon may be behind that one with a different UTR behind it so skipping that exon and going for the alternative last one gives a different (e.g. longer) UTR which can affect that RNA's turnover

Answer 3

normal splice site altered - causes exon skipping as that exon is not cut off from the introns normal splice site altered and another cryptic splice (sequence with potential for interacting with spliceosome) site is used - can e.g. extend an exon mutation causing a new splice site extra exons inserted between existing ones

Answer 4

microRNAs class of ncRNA Pol II transcripts - processed through series of steps in nucleus then cytoplasm end up with ~22 nt mature sequence that guides a protein complex to mRNAs important for regulating mRNA translation and stability

Answer 5

transcribed - then form stem loop structure based on self complementarity recognised by enzymes in nucleus (DROSHA, DGCRA) which cut the stem loop to produce smaller 60-70nt precursor RNA exported to cytoplasm - further processed by DICER to 22nt RNA and on eof these strands is kept because of the stem loop structure of the original transcript - it is still duplex RNA this duplex now binds to Argonaut and one of the strands is kept in this RNP complex - forms and RNA induced silencing complex the 22nt RNA guides the argonaut complex to mRNA (through bp complementarity) to regulate it forms a RIS complex can be created with RNA from introns and exons

Answer 6

in tandem with splicing

Answer 7

homeostasis and quality control (some rnas are transcription by-products need to be removed and recycled, or if the RNA was not made properly) gene regulation (need to get rid of RNA to turn of expression of protein)

Answer 8

exonuclease - degrades from ends require removal of 5' cap to recognise that end (5'-3' activity) for 3'-5' activity - exosome - 6 subunit complex (Rrp6, Rrp44 active nucelases) involved in processsing and degradation recognises abberant RNAs in nucleus endonuclease - cuts internally

Answer 9

most rna degraded in nucleus - either after or during synthesis capping failure - degraded before elongation occurs during splicing: introns need to be degraded too by Dbr1 (after splicing, lariat is debranched and degraded) after termination: RNA transcript aborts (after cleavage of mRNA, "torpedo model") - Rat1 chases polymerase, pol then released export failure - decapping and degradation

Answer 10

detecting aberrant RNA: after ribosome binding: -premature stop codon nonsense mediated degradation -no stop codon non stop decay - when ribosome reads all the way to ribosome tail -stalled ribosome no go decay 3'-5' decay: polyA tail removed by Caf1-Ccr4-NOT and degradation by exosome 5'-3' decay: decapping by Dcp1-Dcp2 and degraded by Xrn1

Answer 11

NMD premature stop codon in mRNA - don't wan early stop protein (nonsense mutation) can happen in long introns that are retained (stop codon sequence UAA can be contained there) recognised as abnormal STOP as normal splicing deposits an Exon junction complex by 5' and 3' splice sites - ribosome kicks them off as it reads - if STOP codon is reached before EJC yet to be kocked -leads to UPF protein recruitment triggerd degradation (decapping, deadenylation - susceptible to exonulceases - Xrn1 and exosome) prevents premature terminated protein from being synthesised

Answer 12

protein only needed for certain time need to degrade its mRNA after a bit remove polyA tail with deadenylase remove 5' cap too - stimulated by PolyA shortening then 5' exonulceases and exosome deadenyilation and decapping linked and mRNA bends so that polyA is in proximity of 5' cap PolyA binding proteins removed by deadenylation interact with cap causing decapping

Answer 13

mRNA half life (normally 15min-10hrs) e.g. cytokines have short half lives and elements in their sequence that mean quick turnover RNA binding proteins (including RNPs MicroRNA-RISC) bind to the 3' UTR downstream of the STOP codon regulate mRNA stability

Answer 14

3' UTR is where degradation regulation happens ARE site mediated: TTP can recognise AU rich elements on the UTR - promotes deadenylation and so degradation of mRNA HUR binds the UTR and prevents TTP from binding - stabilising the mRNA miRNA mediated: RIS complex e.g. argonaut proteins can recognise these sites directly by AA interactions with AU rich sites or through RNA intermediate to complementarity UTR sites (RNPs, RIS complex containing argonaut protein and promotes deadenylation e.g.)

Answer 15

ARE - binding site AUBP - AU binding protein mRNAs with short half lives have AREs in their UTR - hence quick turnover

Answer 16

figuring out of downregulation of mRNA is at trasncriptional level or half life level reporter assay: can see if certain gene is still being transcribed by cloning its promoter to a reporter enzyme (e.g. luciferase, GFP, LacZ) (can identify promoter constructs by going upstream of genes and causing mutations - see if that has an effect on promotion? - increased expression = repressive region was mutated, decreased = promoter mutated) transfect this plasmid vector into cell incubate for RNA and protein to be made measure activity of reporter to see if gene is still being transcribed can measure mRNA degradation activity by cloning the UTR regulatory elements onto plasmid vector downstream of constitutive promoter and reporter gene then transfect this into cell and incubate then stop trasncription with inhibitors (e.g. actinomycin D which intercalates DNA and blocks transcription and elongation) then can measure how long the reporter activity lasts as the mRNA is degraded the activity will drop so can tell whether drop in gene expression is due to transcriptional repression, or is it is due to increased degradation of the mRNA