lecture 19- RNA processing part 2

Question 1

review mRNA processing in eukaryotes

Answer 1

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

Question 2

what can be caused by aberrant pre-mRNA splicing

Answer 2

Duchenne muscular dystrophy and cystic fibrosis

Question 3

are introns trash?

Answer 3

no, they are important for gene transcription

Question 4

number of introns in a gene =

Answer 4

number of exons - 1

Question 5

all pre-mRNA splicing mechanisms consist of the…

Answer 5

ordered breaking and joining of specific phosphodiester bonds to achieve the precise excision of introns

Question 6

rate of splicing in mRNA

Answer 6

splicing must be carried out quickly and correctly to produce the mRNAs required for protein production

Question 7

does mRNA pair with DNA where intron sequences are?

Answer 7

no, b/c the mRNA no longer has introns since they were spliced out so they could become a mature mRNA

Question 8

describe splice junctions for mRNA

Answer 8

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)

Question 9

accurate and efficient splicing in mRNA depends on…

Answer 9

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

Question 10

surrounding sequences of splice junctions

Answer 10

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

Question 11

describe the splice reaction in mRNA

Answer 11

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

Question 12

splice reactions in mRNA are catalyzed by ___

Answer 12

RNPs

Question 13

what catalyzes most pre-mRNA splicing

Answer 13

spliceosome

Question 14

describe structure of spliceosome

Answer 14

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)

Question 15

function of spliceosome

Answer 15

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

Question 16

in the spliceosome, splicing relies on many specific intermolecular interactions, like…

Answer 16

protein-protein
RNA-protein
RNA-RNA

Question 17

describe the 4 steps of the splicing process

Answer 17

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

Question 18

describe self-splicing

Answer 18

• 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

Question 19

describe group I introns in self-splicing

Answer 19

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

Question 20

describe group II introns in self-splicing

Answer 20

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

Question 21

splicing mechanisms and requirements

Answer 21

• 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

Question 22

coordination of transcription and pre-mRNA processing

Answer 22

intron removal is happening at same time as transcription

Question 23

some introns are capable of self-splicing without assistance from any proteins, both kinds, groups I and II, also occur in ___

Answer 23

bacterial and mitochondrial RNA’s

Question 24

ribozymes are enzymes consisting of ___

Answer 24

RNA