Question

A eukaryotic protein-encoding gene contains two introns and three exons: exon 1-intron 1-exon 2-intron 2-exon 3. The 5' splice site at the boundary between exon 2 and intron 2 has been eliminated by a small deletion in the gene. Describe how the pre-mRNA encoded by this mutant gene will be spliced. Indicate which introns and exons will be found in the mRNA after splicing occurs.

Short answer

The pre-mRNA encoded by this mutant gene will be spliced such that the final mRNA product will only include exon 1 and exon 3. The deletion of the 5' splice site at the boundary between exon 2 and intron 2 would likely cause exon 2 to be mistakenly recognized as part of intron 2 and thus excluded along with intron 2 during the splicing process. Therefore, intron 1, exon 2, and intron 2 won't be present in the post-splicing mRNA.

Step-by-step solution

Understanding of gene splicing

Splicing is the process where introns are removed from the pre-mRNA and exons are joined together for the mRNA to be produced. In normal eukaryotic protein-encoding genes, exons are the expressed sequences that will form the final protein. Introns, which are intervening sequences, are removed during mRNA splicing. This allows the exons to be stitched together.

Understand the effect of the 5' splice site deletion

The deletion of the 5' splice site at the boundary between exon 2 and intron 2 means that the machinery that conducts splicing will not be able to recognize the boundary between this exon and intron. So, during the splicing process, the inability to distinguish this boundary may lead to exon 2 being retained as part of the intron, and not included in the final mRNA product.

Predict the mRNA composition after splicing

After splicing of this mutant gene, the mRNA should contain exon 1 and exon 3. The spliceosome will still recognize the boundaries between exon 1 and intron 1 and between intron 2 and exon 3. Exon 2 will likely be spliced out of the final product along with intron 2 due to the unrecognizable 5' splice site at the boundary. Therefore, intron 1, exon 2 and intron 2 will be excluded during the splicing process.

Key concepts

Pre-mRNA Splicing

Pre-mRNA splicing is a critical step in the process that converts the initial RNA transcript synthesized from DNA, known as pre-mRNA, into a mature messenger RNA (mRNA) molecule that can be translated into protein. This process is essential in eukaryotic cells, where the genes contain non-coding sequences called introns, interspersed with the coding sequences, the exons.

During splicing, the cell's molecular machinery identifies specific sequences at the ends of introns and precisely removes the introns, then joins the exons together. The splicing process is essential for producing a variety of proteins from a single gene through a mechanism called alternative splicing, where different combinations of exons can be stitched together to produce different mRNAs and, thus, different proteins.

Exon and Intron

In the context of eukaryotic genes, exons and introns play vastly different roles. Exons are segments within the gene that code for amino acids, and they are conserved in the final mRNA, eventually determining the protein structure. Introns, on the other hand, do not encode protein sequences. Instead, they often contain regulatory elements or can be involved in the regulation of gene expression or mRNA stability.

Introns are removed during the splicing process, which simplifies the initially complex and lengthy pre-mRNA molecule into a mature mRNA with continuous coding sequence. The precise removal of introns and joining of exons is paramount for the correct formation of functional proteins.

Spliceosome

The spliceosome is a large and complex molecular machine in the cell, composed of snRNPs (small nuclear ribonucleoproteins) and various other proteins. It is responsible for carrying out the splicing of pre-mRNA by recognizing and interacting with specific splice sites, the 5' and 3' ends of an intron.

The spliceosome assembles onto the pre-mRNA and goes through several steps - it first identifies the splice sites, then cuts at the 5' splice site, forms a loop called a lariat with the intron, and finally cleaves at the 3' splice site to release the intron and ligate the two exons. This dynamic and regulated assembly ensures that the mRNA is correctly spliced, crucial for producing functional protein products.

5' Splice Site Deletion

A 5' splice site deletion refers to a mutation that removes or alters the sequence at the 5' end of an intron where the spliceosome normally binds to start the splicing process. Such a deletion can have drastic effects on mRNA processing. Without a proper 5' splice site, the spliceosome is unable to recognize the boundary between an exon and an intron accurately.

This can lead to the exclusion of an exon from the mature mRNA, as seen in our exercise where exon 2 is likely to be omitted from the final mRNA product. A deletion at a splice site is a type of splicing mutation that can disrupt the normal functioning of genes and lead to diseases or developmental issues by hindering the production of functional proteins.