14.9: RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded together.

Splicing is Mediated by the Spliceosome

Splicing occurs in the nucleosome and is mediated by a complex of proteins and small RNAs called small nuclear ribonucleoproteins (snRNPs). snRNPs, together with other proteins, form the spliceosome, which recognizes specific nucleotide sequences at the ends of the exon and intron. First, it binds to a GU-containing sequence at the 5' end of the intron and to a branch point sequence containing an A towards the 3' end of the intron. In a number of carefully-orchestrated steps, other snRNPs then bring the branch point close to the 5' splice site. Subsequently, a chemical reaction cleaves the 5' end of the intron from its upstream exon and attaches it to the branch point, forming a loop called a lariat. To release the lariat, the AG-containing sequence of the intron near the 5' end of the downstream exon reacts with the 3' end of the upstream exon. This reaction patches the two exons together, concluding the splicing process.

Splicing Allows the Expression of Several Proteins from a Single Gene

Typically, exons are joined together in the order in which they appear in a gene. However, during alternative splicing, different combinations of exons in pre-mRNA are combined to form mature mRNA. This produces several different proteins from a single pre-mRNA transcript.

Different patterns of alternative splicing include exon skipping, alternative 5' or 3' splice sites, and intron retention. These patterns are guided by the length of exons or introns and the strength of the splicing signal at the splice sites. Because of this, exons that are shorter than other exons may be overlooked by the spliceosome and omitted from the mature mRNA. In contrast, introns that are significantly shorter than other introns may evade removal by the spliceosome and are retained in the mature mRNA. As a result, alternative splicing generates variants of mature mRNA that were copied from the same stretch of DNA. The RNA sequence variants produce different proteins with additional or fewer amino acids, shifts in the reading frame, or a premature stop codon. These protein isoforms have different biological properties, including function, cellular localization, and interaction with other proteins, thereby playing a vital role in tissue- and environment-specific gene expression.

Abnormal Splicing Can Cause Diseases

Errors in splicing can produce aberrant protein isoforms, which may contribute to diseases, including cancer. For instance, alternative splicing of the BCL2L1 gene generates a long and short protein isoform—BCL-XL and BCL-XS, respectively— through the use of alternative 5' splice sites. The longer BCL-XL isoform promotes cell survival and is highly expressed in several types of cancers (e.g., blood, breast, and liver cancers). Expression of the short BCL-XS isoform that promotes cell death is suppressed in cancer.

When pre-mRNA is transcribed from DNA, it contains exons, protein-coding sequences, and introns, non-coding regions. RNA splicing removes the introns and attaches the exons together.

This is catalyzed at the spliceosome, a large assembly of small nuclear ribonucleoproteins, or snRNPs — complexes made up of small nuclear RNAs and proteins.

The spliceosome is composed of 5 snRNPs, U1, U2, U4, U5 and U6, and several other proteins.

First, U1 snRNP binds the 5' splice site while the branch point sequence containing A is recognized by other proteins and later replaced by U2 snRNP. 

Next, a complex of U4/U6 and U5 snRNPs binds to the 5' splice junction and assists in bringing the 5’ end to the branch point A. A transesterification reaction between the branch point A and the 5' splice site creates a loop called a lariat.

Following this, another transesterification reaction occurs between the 3' end of the upstream exon and the 5' end of the next exon. This cleaves off the lariat containing the intron, leaving the exons attached to each other.