lecture 19- RNA processing part 2 Flashcards
review mRNA processing in eukaryotes
transcription by RNA poly II
- addition of 5’ cap when 20-30 nucleotides of pre-mRNA are made
- addition of 3’ polyA tail
- RNA splicing to remove introns
what can be caused by aberrant pre-mRNA splicing
Duchenne muscular dystrophy and cystic fibrosis
are introns trash?
no, they are important for gene transcription
number of introns in a gene =
number of exons - 1
all pre-mRNA splicing mechanisms consist of the…
ordered breaking and joining of specific phosphodiester bonds to achieve the precise excision of introns
rate of splicing in mRNA
splicing must be carried out quickly and correctly to produce the mRNAs required for protein production
does mRNA pair with DNA where intron sequences are?
no, b/c the mRNA no longer has introns since they were spliced out so they could become a mature mRNA
describe splice junctions for mRNA
sequences within mRNA determine where splicing occurs
- 5’ splice site is “always” GU and 3’ splice site is “always” AG
- so that the generic intron would have the sequence: GU… …AG (minority of introns have AU…AC)
accurate and efficient splicing in mRNA depends on…
relies on base pairing between the pre-mRNA and snRNA’s of spliceosome to specify the bonds to be broken or formed
- sequential assembly and rearrangement of spliceosome on the intron to be removed
surrounding sequences of splice junctions
a branch point in the right context is 20-50 nucleotides upstream of the 3’ splice site
- the surrounding sequences and perhaps the structure of the pre-mRNA itself must play a role in the selection of splice sites
describe the splice reaction in mRNA
two site-specific transesterification reactions resulting in phosphodiester bond cleavage and ligation
- formation of lariat (lasso, loop, and tail structure
- intron is released and exons are joined together
- splicing requirements: splice site and the branch point site
- splice reactions are catalyzed by ribonucleoproteins (RNP’s): complexes of non-protein-coding RNA’s and proteins
splice reactions in mRNA are catalyzed by ___
RNPs
what catalyzes most pre-mRNA splicing
spliceosome
describe structure of spliceosome
large complex of 5 small nuclear ribonucleoproteins (snRNPs - snurps) + hundreds of additional protein components
- single small nuclear RNAs (100-300 nt) = snRNA’s (U1, U2, U4, U5, U6) in each snurp
- “snurps” have a name corresponding to snRNA that they carry
- each snRNA is complexed with several (under 20) proteins that forming snRNPs (snurps)
function of spliceosome
snRNPS’s (with help from proteins) have several functions:
- recognition of 5’ and 3’ splice sites and the branch site bringing those sites together; and catalysis of cleavage and joining reactions
in the spliceosome, splicing relies on many specific intermolecular interactions, like…
protein-protein
RNA-protein
RNA-RNA
describe the 4 steps of the splicing process
1- U1 binds to the 5’ splice site; U2 binds to the branch point
2- the U4-U5-U6 trimeric snRNP displaces U1 at the 5’ splice site, then U4 dissociates
3- U6 and U2 catalyze attack of the branch point on the 5’ splice site
4- 5’ splice site attacks the 3’ splice site, completing the reaction
describe self-splicing
- trancripts other then nuclear pre-mRNA may contain introns that will be spliced
- all non-nuclear transcripts do not use spliceosome
- they are self-spliced and the catalysis is performed by the intron itself (ribozyme) - no involvement of any protein enzymes
- these transcripts belong to 2 classes: group I and group II based on the mechanism of the 1st transesterification reaction
describe group I introns in self-splicing
rare; nuclear rRNA in some eukaryotes, organelle genes, and a few prokaryotic genes
- two transesterification reactions, requires a guanine nucleoside or nucleotide cofactor (not used as a source of energy) for the 1st transesterification reaction
- catalytic machinery = ribozyme
- the 3’ -OH of guanosine acts as a nucleophile, attacking the phosphate at the 5’ splice site —> the 3’-OH of the 5’ exon becomes the nucleophile, completing the reaction
- ends in linear form
describe group II introns in self-splicing
rare: some eukaryotic genes from organelles and prokaryotes
- mechanism is same as pre-mRNA
- catalytic machinery = ribozyme
- the structure of the RNA itself, rather than the assembly of multiple snRNP’s, creates an active site for catalysis
- similar to spliceosome, but introns present catalytic activity so it doesn’t need a spliceosome
splicing mechanisms and requirements
- group I and group II introns self-splice
- the information for splicing includes catalytic activity is present in group I II introns
- all three classes of splicing reactions proceed by two transesterifications
- most of the self-splicing intron sequence is critical
coordination of transcription and pre-mRNA processing
intron removal is happening at same time as transcription
some introns are capable of self-splicing without assistance from any proteins, both kinds, groups I and II, also occur in ___
bacterial and mitochondrial RNA’s
ribozymes are enzymes consisting of ___
RNA
