2. RNA Processing Flashcards
describe the steps of 3’ cleavage and polyadenylation
1) poly A polymerase (PAP) and cleavage proteins recognize poly A signals : AAUAAA and a G/U signal which are upstream of the 3’
2) the complex recruitment results in cleavage of the precursor mRNA 10-35 nucleotides from the 3’ end
3) PAP adds 12 A residues slowly to the 3’ cleaved ends (slowly)
4) Poly A Binding Protein (PABPII) associates and allows PAP to add 200-250 more residues (fast- kicks it into high gear)
describe the steps of 5’ capping for mRNA processing
- the terminal residue has a 5’ triphosphate. One phosphate is removed by phosphohydrolase
- Then a guanylyl transferase adds GTP in a 5’ to 5’ (unusual linkage)
- 5’ cap is further modified by addition of several methyl groups (keeps protein from being eaten up)
why are 5’ and 3’ modifications important? when do they occur?
they protect again degradation
the 5’ cap actually
1) protects against degradation by exonucleases
2) important for mRNA splicing
3) position the ribosome on the mRNA for translation
they actually occur while the protein is being translated, so after about 25 bp
what are the processing activities for messenger RNA?
1) 5’ capping
2) 3’cleavage and polyadenylation
3) splicing
4) RNA editing
why is the poly A tail and associated PABPs so important
they
1) block 3’ degradation of the mRNA by exonucleases
2) essential for mRNA export from nucleus to cytoplasm
when do mRNAs get degraded
over time the poly A tail shortens and when it reaches crucial length of 30 A, then get rapid degradation to clear it out of the cell.
degradation occurs by:
-decapping
-and then 5’ to 3’ degradation and 3’ to 5’ degradation
what happens in splicing? give overview
in splicing, the introns or intervening sequences are removed and the exons or expressed sequences are rejoined
___ size tends to be more uniform than ___ although the number of exons and introns within a gene can vary
exon size tends to be more uniform than intron length (think of it as introns are intervening randomly)
describe WHERE the cell knows to cut the introns and exons
there are consensus sequences at both the 5’ and 3’ ends that indicate splicing:
1) GU right after 5’ splice site
and
2) AG right before 3’ splice site
3) there is also a conserved branch point A
describe the transesterification during splicing. what is released at the end?
1) the 2’OH hydroxyl group from the A residue in the branch site attacks the phosphodiester bond at the 5’ splice site
2) the 3’OH generated at the end of the axon then attacks the phosphodiester bond at the 3’ splice site
3) results in joining of the 2 exon sequences and intron released as a lariat molecule
describe the mRNA spliceosomes. how is the positioning key?
it is a RNA/protein complex that includes the pre-mRNA and five small nuclear RNAs called snRNAs
(also included are U1, U2, U4, U5, and U6
also have about 100 proteins known as splicing factors which we don’t have to know.)
they actual do complementary base pairing in order to position the sequences to be spliced and catalyze the reaction
*this positioning is key and uses ATP
what about splicing in other animals? what does this mean? what evidence implicates this
-they do self splicing so they don’t require other proteins or co-factors for RNA splicing
Group 1 introns: in mRNA, tRNA, and rRNA of mitochondria and chloroplasts. also in bacteriphages and eubacterial
group 2 introns: found in mitochondria of fungi and chlorplasts of plants. Also cyanobacteria and proteobacteria.
- this resembles that of eukaryotic, hinting to evolutionary preservation of mechanism
- also there are structural similarities btwn pr-mRNA spliceosome and Group II introns that implicate its evolutionary origin
describe the importance of alternative splicing and give an example
describe all the other mechanisms that yield diversity in mRNA transcripts
transcripts of 75% of genes are spliced in more than one way.
this is a huge reason for diversity in proteins this is a conscious effort of the cell and not a mistake
example given was tropomyosin- actin binding protein that regulates actin mechanics. It can be cut in many different ways to yield: striated muscle, smooth muscle, fibroblast, brain mRNA
1) alternate splicing:
2) alternate promoters
3) alternate polyadenylation sites
what mistakes can happen with splicing? give a clinical example
so with splicing we can have splicing in incorrect places and this will yield more or less introns/exons.
example given is beta thalassemia which is a blood disorder of excessive RBC loss and severe anemia
how does RNA editing work? what is the difference in where the editing occurs? what enzyme is important? how are they important medically?
nucleotide sequence is altered so here the sequence of the resulting mRNA differs from the genomic DNA sequence
this can affect coding AND non coding sequences
-they help tell enzymes where to insert new nucleotides into mRNA transcript in MITOCHONDRIA
editing in non-coding regions of pre-mRNA can lead to alternative splicing or affect the translation, transport, or stability of the mRNA transcript
- the trypanosome gene inserts Uracil nucleotides and this changes the meaning of the message
- guide RNAs have 40-80 nt RNA sequences that specify where to insert the U nucleotides into the pre-mRNA transcript
- enzyme uridyly transferase adds the Us
drugs to inhibit this complex is promising for therapy for those suffering from sleeping sickness or chagas disease
