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Overview

The process in which eukaryotic RNA is edited prior to protein translation is called splicing. It removes regions that do not code for proteins and patches the protein-coding regions together. Splicing also allows several protein variants to be expressed from a single gene and plays an essential role in development, tissue differentiation, and adaptation to environmental stress. Errors in splicing can lead to diseases such as cancer.

RNA Transcribed from Eukaryotic DNA Undergoes Several Modifications

The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts designated to become mRNA are called precursor messenger RNA (pre-mRNA). The pre-mRNA is then processed to form mature mRNA that is suitable for protein translation. 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. RNA splicing is the process by which introns are removed and exons patched together.

Splicing Occurs within the Nucleus

Splicing is mediated by the spliceosome—a complex of proteins and RNA called small nuclear ribonucleoproteins (snRNPs). The spliceosome recognizes specific nucleotide sequences at exon/intron boundaries. 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 3’ end of the upstream exon reacts with the AG-containing sequence of the intron close to the 5’ end of the downstream exon. This reaction patches the two exons together and, thus, concludes the splicing process.

Splicing Allows Expression of Several Proteins from a Single Gene

The process by which different combinations of exons in pre-mRNA are joined to form mature mRNA is called alternative splicing. Alternative splicing produces several different proteins from a single pre-mRNA transcript.

Normally, exons are joined together in the order in which they appear in a gene. However, during alternative splicing, this preferred sequence of exons may be altered. 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 splice sites. Consequently, 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.

The strength of splice sites is determined by sequence conservation around alternative exons; these influence the 5’ or 3’ splice sites chosen by the spliceosome. Thus, alternative splicing generates variants of mature mRNA that were copied from the same stretch of DNA.

During translation, the RNA sequence variants produce different proteins with additional or fewer amino acids, shifts in the reading frame, or a premature stop codon. This generates protein isoforms with different biological properties including function, cellular localization, and interaction with other proteins. Alternative splicing plays a vital role in gene expression, thereby controlling organ development, cell survival or proliferation, and adaptation to environmental changes.

Abnormal Splicing Can Cause Diseases

Errors in splicing may be caused by mutations in the gene itself or in the regulatory elements that control the expression of the gene. A mutation that occurs in the exon or intron sequence of a specific gene transcript is called a cis-mutation. A mutation in the splicing machinery affects several genes and is called a trans-mutation.

Errors in splicing 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-X_L and BCL-X_S, respectively—through the use of alternative 5’ splice sites. The longer BCL-X_L 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-X_S isoform that promotes cell death, is suppressed in cancer.