Splicing

Question 1

What is splicing?

Answer 1

In eukaryotes, transcription produces precursor mRNA (pre-mRNA), which comprises the majority of heterogeneous nuclear RNA (hnRNA) in the nucleus. Pre-mRNA is processed in the nucleus to form mature RNA following 5’ capping, 3’ polyadenylation and finally splicing.

Splicing removes introns by endonucleolytic cleavage and ligation of exons

Question 2

What are splice donor and splice acceptor sites?

Answer 2

Most introns start from the sequence GT (or GU at the RNA level) and end with the sequence AG (in the 5’ to 3’ direction). They represent the splice donor and splice acceptor site, respectively.

However, the sequences at the two sites are not sufficient to signal the presence of an intron. Another important sequence is called the branch site located ~20 - 50 bases upstream of the acceptor site. The branch point always contains an adenine, but it is otherwise loosely conserved

Question 3

What is the splicing mechanism?

Answer 3

In short, it involves five snRNAs and their associated proteins. These ribonucleoproteins form a large (60S) complex, called spliceosome. After a two-step enzymatic reaction, the intron is removed and two neighbouring exons are joined together. The branch point A residue plays a critical role in the enzymatic reaction.

Processivity of the spliceosome is largely progressive in a 5’ to 3’ direction in one intron (i.e. a 5’ splice site is recognised and then it scans until it meets a 3’ site) although introns are not necessarily spliced out in order (e.g. intron 1, then intron 2); this is determined by RNA 20 structure and how accessible they are to the splicing machinery.

Question 4

What is the spliceosome?

Answer 4

Splicing reactions are mediated by the major (U2 dependant) and minor (U12 dependant) spliceosome, complexes of small nuclear RN A (snRNA) molecules (most important U1, U2, U4, U5 and U6 in the major and U11, U12, U4atac, U5 and U6atac in the minor spliceosome) and a vast range of proteins (>100 apparently involved in splicing mechanism).

Question 5

Sequential steps of pre-mRNA interactions with the snRNA complex

Answer 5

1. U1 snRNA binds to donor splice site (“GU”) (via its complementary sequence: UACUUAC)
2. U2 snRNA binds to branch site (“A” nucleotide), U1 and U2 come together forming a loop in the pre-mRNA
3. U4, U5 & U6 associate with U1-U2 to form the spliceosome;
4. The bonding of the 5’ guanine and the branchpoint adenine bases takes place via a chemical reaction known as transesterification (Nucleophilic attack of GU (donor) by 2’ hydroxyl group of A of branch point). This forms lariat.
5. U5 binds both donor & acceptor splice sites. Cleavage of acceptor (“AG”) by transesterification #2 joins exons together
6. Lariat intron & spliceosome unbound

Question 6

How is splicing regulated?

Answer 6

Splicing, in addition to the efficacy of the consensus sites, is regulated by trans-acting elements (proteins translated from other genes with both specific and general targets) and corresponding cis-acting elements (regulatory sites, both intronic and exonic, within the RNA) to which they bind.

Question 7

How is splicing regulation controlled?

Answer 7

Regulation is tissue- (cell chemistry), context- (presence/interaction of other trans-acting elements) and timing- (cell cycle/current cell role) specific, and controlled by a cascade of transcripts from other genes.

Question 8

What is an exonic splicing silencer (ESS)?

Answer 8

A short region (usually 4-18 nucleotides) of an exon and is a cis-regulatory element.

ESSs inhibit or silence splicing of the pre-mRNA and contribute to constitutive and alternate splicing. To elicit the silencing affect, ESSs recruit proteins that will negatively affect the core splicing machinery. Exonic splicing silencers work by inhibiting the splicing of pre-mRNA strands or promoting exon skipping.