RNA processing Flashcards
Why is the lack 2’ hydroxyl group on the DNA structure important?
It is lacking, so DNA is UNSTABLE
Describe the differences In PROKARYOTES and eukaryotes.
PROKARYOTES: mRNA is a direct copy of the DNA gene sequence.
Most genes in higher eukaryotes are SPLIT and have their protein-coding information interrupted by non-coding, ‘intervening’ sequences, called “introns” which are included in mRNA PRECURSOR.
THESE SEQUENCES ARE REMOVED BY CLEAVAGE and SPLICING and POLYADENYLAION.
The sequences ‘expressed’ in mRNA are called “exons” make the final mRNA
.
Prokaryotes don’t need splicing to make correct proteins; eukaryotes do.
Explain the problem of introns.
In almost all genes in multicelled eukaryotes: The introns often contain STOP codons that would PREMATURELY terminate translation or the introns may alter the READING FRAME FOR EXONS
Therefore RNA splicing must occur or cells to make the correct proteins needed; ALL INTRONS ARE REMOVED
How is RNA spliced in complex organisms?
Multiple introns may be spliced differently in different circumstances, for example in different tissues.
This because some genes are unique: same RNA sequence can act as an intron in one transcript and as an exon in another transcript.
What is snoRNA?
Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that primarily guide chemical modifications of other RNAs; they are present in pre-mRNA.
The two main classes of snoRNA with different structures and conserved residues.
1) the C/D box snoRNAs, which are associated with methylation,
2) H/ACA box snoRNAs, which are associated with pseudouridylation.
How does snoSNA function?
Each snoRNA molecule acts as a guide for only one/two) individual modifications in a target RNA.
snoRNA associates with at least four protein molecules (small nucleolar ribonucleoprotein (snoRNP) in an RNA/protein complex .The proteins associated with each RNA depend on the type of snoRNA molecule family.
The snoRNA molecule contains an antisense element which is base complementary to the sequence surrounding the base targeted for modification in the pre-RNA molecule.so snoRNP can recognise and bind to the target RNA.
Once the snoRNP has bound to the target site, the associated proteins are in the correct physical location to catalyse the chemical modification of the target base
What is known to happen to snoRNA and miRNA in terms of splicing
Most snoRNAs and a large fraction of miRNAs are encoded in introns of RNA polymerase II transcripts which is spliced out of the sequence despite being FUNCTIONAL PRODUCTS.
What is a spliceosome?
A complex of 300 proteins and 5 small RNAs that is responsible for catalysing the splicing of introns from pre-mRNAs.
It recognises sequences through a SPLICE SITE CONSENSUS SEQUENCE at either end of the intron (5’ and 3’)
A major splicesome is one that removes the same general structure of most introns, however, some introns are unique and are removed by the MINOR splicesome
Why are some introns removed by the Minor splicesome?
These introns splice sequences differ and have been termed AT-AC.
How do spliceosomes recognise the splice sites? What evidence is here for this?
the major sub-units snRNPs which interact with bases of the intron branch point (middle of intron) and the end of the intron base (via the trans-acting components)
U1 snRNA base pairs with end of intron
U2snRNA base pairs with the branchpoint
Evidence for base pairing between snRNAs
and intron sequences:
1)phylogenetic conservation 2)biochemical RNA X-linking data
3)genetic compensatory mutation studies
Describe the two steps involved in Splicing.
The spliceosome first makes a LARIAT intron still attached to one exon. In this step there is a ‘trans-esterification’ where the 2’ hydroxyl group of the branch site A residue attacks the phosphodiester bond between exon 1 and the 5’ end of the intron.
Spliceosome then catalyses another ‘trans-esterification’ where the 3’ hydroxyl group
of the FREE exon 1 attacks the phosphodiester bond between the 3’ end of the intron and the exon 2
Describe the splicesome cycle.
a series of intermediate complexes as the separate snRNP subunits and additional protein splicing factors are recruited onto the pre-mRNA.
The assembly of spliceosomes occurs on nascent pre-mRNA in vivo - introns can be removed before the RNA polymerase has completed transcription of the gene
Spliceosomes do NOT move along the pre-mRNA removing each intron in turn; introns treated individually by separate spliceocomes.
What is meant by an ISOENERGETIC splicing reaction?
The reaction during splicing cause NO overall change in the NUMBER of chemical BONDS formed or broken and therefore NO ENERGY is needed for these reactions
But energy is needed in the assembly of the spliceosome itself.
Describe alternative splicing
It increases as species complexity increases, and is the main way protein ISOFORMS are formed; nearly all of the genes have protein isoforms via alternative splicing. (95%)
List Alternative splicing patterns.
1) Exon skipping (an exon excluded too)
2) Mutually exclusive exons(different exons included)
3) alternative 3’ss(first parts of exons skipped)
4) alternative 5’ss (end part of exon skipped
5) exitrons (middle chunk of extron excluded)
6) alternative promoters (different combinations of extrons)
7) alternative polyadenylation (different exons have the AAA tail)
8) intron retention (intron excluded only)
