lec 7.5 - RNA splicing

why do eukaryotes have introns? advantages?

• alternative splicing of them allows for more proteins to be made

mRNA is modified in the ______

• how is it modified?

nucleus

1. addition of cap to the 5ʹ end

2. splicing to remove the introns

   • requires breakage of the exon–intron junctions and joining of the ends of the exons

3. addition of A’s to 3ʹ end

4. RNA editing

what determines which introns are being spliced out?

• expression of diff. small nuclear ribonucleoproteins (snRNPs) in diff cells and at diff times regulate which introns are being spliced

what happens to mature mRNA

• it gets transported through nuclear pores cytoplasm where it gets translated

explain the addition of cap

• add 7-methylguanosine (7-meG) - the 5’ cap

  • G is methylated

• attachment is 5’ to 5’ phosphodiester bond

• means added backwards

functions of 5’ cap

• adds to stability of RNA

• used as identity marker for ribosome to recognize

capping enzyme

• enzyme - guanyltransferase

• attached to CTD domain (phosphorylated) of pol II to be sure every RNA transcribed by RNA pol II is capped

how do we know 5’ cap is necessary?

• experiment showed that capped mRNA gets bound to ribosome

• uncapped mRNA does not bind ribosomes

• therefore the 5’cap is necessary for ribosome binding

how do we know there are introns in DNA?

• hybridized mRNA and corresponding DNA sequence has loops

• e^- microscopy shows loops which indicate where the introns are

GU-AG Rule (U2 Type)

• seen in majority of introns

• defined the requirement for these constant dinucleotides at the first 2 and last 2 positions in the intron

• the ends of nuclear introns are defined by the GU-AU rule (seen as GT-AG in the DNA sequence)

AU-AC Rule (U12 Type)

• observed in a minority of introns

• minor introns are defined by different consensus sequences at the 5’ splice site, branch site and 3’ splice site

what do these rules ensure

accurate and regulated splicing of pre-mRNA, allowing for generation of mature mRNA molecules

splicing occurs in two stages, describe them

1. 5ʹ exon is cleaved off, then it is joined to the 3ʹ exon

2. adenine (A) serves as branch point

• nuclear splicing occurs by two transesterification reactions, in which an -OH group attacks a phosphodiester bond

  • carried out by spliceosome

  • intron gets released

what do spliceosomes recognized?

• 5’ splice site

• branch site

• 3’ splice site

spliceosome composition

• approximately 12 megadaltons (MDa)

• complex of RNA and proteins

• five snRNPs account for almost half of mass

• remaining proteins include known splicing factors and proteins involved in other stages of gene expression

how many snRNPs in spliceosomes

• 5 snRNPs (snurps)

  • U1, U2, U4, U5 and U6 (not for memory)

• plus hundreds of additional proteins

• AND one small nuclear RNA (snRNA) at the heart of each snurp

U1 small nuclear RNA (snRNA)

• describe the structure

• base-paired structure that creates several domains.

• 5ʹ end remains single stranded and can base pair w/ 5’ splice site

U1 in splicing - step 1

• U1 binds to GU at 5’ splice site and U2AF binds to AG at 3’ site and helps U2 bind to branch point (A)

• requires energy (ATP hydrolysis)

U1 in splicing - step 2

• 2’OH at branch point then becomes a nucleophile that attacks the phosphodiester bond at the 5’ splice site

• U4-U6-U5 trimeric snRNP complex displaces U1 at the 5’s splice site then U4 dissociates

• U1 and U4 snRPS are released

U1 in splicing - step 3

• U6 and U2 catalyze n-philic attack of 2’OH branch point A on phosphodiester bond at the 5’ end – cleaving the 5’ exon- intron junction

• this causes U5 to shift to 3’ splice point

• first step in transesterification

U1 in splicing - step 4

• results in lariat (intron that’s cut out in a lasso/non-linear shape) containing the intron attached by the 3’ end

• U2-U6-U5 remains attached and causes nucleophile attack on phosphodiester bond at 3’end

• results in release of intron and joining of 5’ and 3’ ends on exons

• requires energy

• second step in transesterification

summary

• splicing reaction proceeds through discrete stages in which spliceosome formation involves the interaction of components that recognize the consensus sequences

describe the experiment that showed that some introns can self-splice (no need for spliceosome)

• eukaryotic gene cloned into bacterial plasmid vector and transcribed with bacterial RNA polymerase in vitro

  • found it had spliced itself (without help from any proteins)

  • reveals that RNA is capable of enzymatic activity

name and describe the 2 classes of self splicing introns

• group I introns

  • always self-splice

  • share a common structure

• group II introns

  • usually use a spliceosome (as a catalyst) but are capable of self-splicing

  • share a common secondary structure

group I introns

• structure

• mechanism

• lot’s of stem loops to bring exons together

• requires a GTP as a co-factor not for energy

• 3’ OH used as nucleophile

  • 3’ OH of guanosine acts as a nucleophile and attacks the phosphate at the 5’ splice site

  • 3’ OH of the 5’ exon becomes the nucleophile and completes the reaction

  • exons are joined

  • intron is spliced out in a linear form

group II introns

• structure

• mechanism

• lot’s of stem loops to bring exons together

• domain I binds to 5’ and 3’ splice sites

• domain VI contains A the functions as a nucleophile

• domain V contains sequences critical for splicing reaction to be efficient

• lariat structure forms

trans-splicing

• occurs in nematodes (conserved in that group)

• short capped leader sequence is spliced onto a pre-mRNA to make a mature mRNA

adding poly A tail

• length

• function

typically 80–250 residues long

• length determines how long mRNA lasts

function:

• stability by blocking access of ribonucleases to 3’ end

mRNA capping, polyadenylation and intron splicing

all three are coordinated