mRNA processing: spliceosomal introns

splicing vs alternative splicing

1. all introns removed and all exons remain in mRNA

2. some exons skipped, rendering different mRNAs from same gene

what composes the spliceosome, generally?

large ribonucleoprotein (RNP) particles which assemble at the 5’ and 3’ splice site regions

when there are two RNP particles on a transcript,

they occur at or very near the positions of the 5’ and 3’ splice cites for the single small intron sequence (228 nucleotides long) near the 5’ end of the transcripts

spliceosome

a large (~45S) particle that excises spliceosomal introns from pre-mRNA

contains 5 RNAs (small nuclear RNAs = snRNAs) and 60-100 proteins

consensus sequences at/near 5’ and 3’ splice sites in vertebrate pre-mRNAs

5’ GU and 3’ AG of intron

pyrimidine (CU)-rich region near 3’ end of intron

essential branch-point adenosine 20-50 residues from 3’ splice site

only invariant residues of splice sites in vertebrate pre-mRNAs

5’ GU and 3’ AG of intron, although flanking residues are found at frequencies higher than expected based on random distribution

which region in vertebrate pre-mRNA is generally unnecessary for splicing?

the central region of the intro, which can be 40 bases to 50 kb in length

pre-mRNA splicing reaction mechanism

1. nucleophilic attack of 5’-P at 5’ intron boundary by 2’-OH of internal A residue (branch point A or bulging A). reaction yields a lariat intermediate, in which the intron forms a loop

2. 3’ OH of released exon 1 attack 5’ terminal phosphate of exon 2, resulting in excision of the intron as a lariat structure; the intro is rapidly degraded

small nuclear RNAs (snRNAs)

relatively abundant, U-rich nuclear RNAs

relatively small (typically ~100-200 nt)

exist as ribonucleoprotein particles → snRNPs

U1 snRNA function

binds the 5’ splice site then the 3’ splice site

U2 snRNA function

binds the branch site and forms part of the catalytic centre

U6 snRNA function

catalyzes splicing, interacts with U2

what do snRNAs interact with?

mRNAs, each other, and protein components of snRNPs

interactions between pre-mRNA, U1 snRNA, and U2 snRNA early in splicing

5’ region of U1 initially base pairs with 5’ end of intron and 3’ end of 5’ exon of pre-mRNA

U2 base pairs with sequence that include branch point A, although this residue is not base-paired

sequences in snRNAs bind certain snRNP proteins called SM proteins

splicing catalytic centre

formed by U2 and U6 snRNA, which are base paired

U2 is also base paired to branch site of mRNA precursor (branch point A residue bulges out)

splicing mechanism overview

snRNAs in the form of snRNPs associate with pre-mRNA in a stepwise fashion to form a splicing complex, the spliceosome

interactions between U1, U2, and mRNA constitute the earliest stages of spliceosome assembly

catalytic centre of spliceosome formed by U2 and U6

2’-OH of internal A residue (branch point) initiates first of two splicing reactions

carboxy-terminal domain (CTD)

the one of the largest subunit of RNA pol II contributes to 3 aspects of mRNA processing

CTD interacts with

enzymes that synthesize 5’ cap and with cap-binding complex (CBC)

the 3’ cleavage and polyadenylation enzyme complex

some spliceosomal components (splicing factors)