Eukaryotic RNA Processing

Unlike prokaryotes, eukaryotes must process mRNAs before they can be translated into proteins. The primary mRNA transcript is first made by transcription of the DNA by RNA pol II. The transcript contains both introns and exons, hence it is called heterogenous RNA (hRNA). The 5′ end of the hRNA is capped and a polyA tail attached to the 3′ end. Splicing of the RNA to remove introns then follow. This last step produces the mature RNA, called messenger RNA (mRNA).

mRNA Capping at the 5′ end

As soon as the hRNA emerges from RNA pol II (after only 20-30 nucleotides are transcribed) the cap is added to the 5′ end of the RNA. This cap is a 7-methyguanine nucleotide attached via an unusual 5′-5′ triphosphate linkage. Adding a 5′ cap achieves the following objectives:

1. Efficient elongation and termination of transcription
2. Facilitates mRNA processing
3. Acts as a binding site for proteins that export mRNA from the nucleus to the cytoplasm
4. Directs initiation of protein synthesis from mRNA
5. Protects the mRNA from degradation by 5′ to 3′ nucleases

Polyadenylation at the 3′ end

The 3′ end of the hRNA is adenylated by an enzyme named polyadenylase with the addition of approximately 200 adenosine nucleotides, forming what is called a polyA tail. The process begins with an initial cleavage of the mRNA at a point after a CA nucleotide pair that lies between a conserved 5’-AAUAAA-3’ sequence and a U or GU-rich region further downstream. The polyA tail is added once the cleavage is complete. Some hRNAs have more than one polyadenylation site. Depending on the selected site of polyadenylation, the hRNA will be longer or shorter. Shorter hRNA will generally mean less available sites for proteins that regulate mRNA stability and translation.

Benefits of polyadenylation include:

1. Efficient termination of transcription
2. Enabling export of mRNA to cytoplasm

RNA Splicing

Introns must be removed from the hRNA tp produce a mature mRNA ready to be translated. Introns may be excised by proteins, by ribonucleoproteins (RNPs), or may excise themselves. After being excised, introns are usually degraded. RNA splicing generally occurs by transesterification reactions. This is a 2-step reaction where the 5′ end of the intron is detached from exon 1, exposing an OH group that is free to react with the other end of the intron. In the process the  intron is excised and exons 1 and 2 are joined.

Group I Self-Splicing Introns

Group I introns are a class of introns that function as ribozymes. This allows them to catalyze their own excision. The first step of transesterification involves the 3′ end of a free exogenous guanosine attacking the 5′ end of the intron. In the second step, the end terminus of exon 1 attacks the intron-exon 2 junction, joining the two exons.

Group II Self-Splicing Introns

Group II introns are larger in size to the group I introns and conduct a different sequence of transesterification reactions. First, the 2′ OH of an adenosine (A) in the intron attacks the exon 1-intron junction where the nucleotides GU is located. Exon 1 then attacks the intron-exon 2 boundary to join exon 1 and 2. The resulting lariat formed between A and U is removed in the process.

RNA Splicing by the Spliceosome

The majority of introns are not self-splicing. These require a ribonucleoprotein machine called a spliceosome. This machinery consists of several small nuclear ribonucleoproteins (snRNPs) (RNA + protein), called “snurps”. The mechanism of splicing is similar to group II introns. However, before splicing can occur, the spliceosome must identify the splice sites i.e., the 5′ splice site, where there is GU, the 3′ splice site, where there is AG, and the A branch point. The main snRNPs that constitute the spliceosome are U1, U2, U4, U5, and U6. Here are the steps in the splicing process using spliceosomes.

1. U1 base pairs with GU at the 5′ splice site
2. U2 base pairs with the A at the branch site
3. The remaining snRNPs (U4, U54 and U6) join U1 and U2 to assemble the spliceosome. In this step, the spliceosome brings the two exon ends close together
4. U1 and U4 are expelled
5. U6 base pairs with the 5′ site and U2
6. The 5′ end of the intron cleaves and then attaches to the A branch point
7. The 3′ end cleave and exons are joined together
8. The intron exits as a lariat and is later degraded by enzymes

Alternative Splicing

A single gene may produce several different protein products depending on where splicing takes place along the hRNA. You can see this from the illustration below in which the letters are the exons and the numbers are the introns.

Alternative splicing may be dependent on such factors as cell type, developmental stage of the organism, and the cellular environment.

Reference: Molecular Biology – Principles of Genome Function (Third Edition) by Nancy Craig; Rachel Green; Carol Greider; Gisela Storz; Cynthia Wolberger. Published by Oxford.