Question

Present an overview of various forms of posttranscriptional processing in eukaryotes. For each, provide an example.

Short answer

Question: Provide an overview of various forms of posttranscriptional processing in eukaryotes and their roles in gene expression. Answer: Posttranscriptional processing is a series of modifications that occur after the transcription of a gene into a pre-mRNA molecule in eukaryotic cells. These modifications generate a mature mRNA that is ready for translation and protects the mRNA from degradation. Some common posttranscriptional modifications include: 1. 5' capping - This adds a 7-methylguanosine cap to the 5' end of the mRNA molecule, protecting it from degradation, assisting in nuclear export, and helping recruit translation machinery. 2. 3' polyadenylation - This adds a poly(A) tail to the 3' end of an mRNA molecule, enhancing mRNA stability, nuclear export, and translation efficiency. 3. RNA splicing - This process removes non-coding introns from the mRNA and joins coding exons together to create a continuous reading frame for protein synthesis. 4. Alternative splicing - This allows a single gene to produce multiple mRNA isoforms through combinatorial use of exons, so that distinct protein isoforms can be generated for various cellular processes.

Step-by-step solution

Introduction to posttranscriptional processing

Posttranscriptional processing refers to a series of events that occur in eukaryotic cells after the transcription of a gene into a molecule of precursor messenger RNA (pre-mRNA). These events modify the pre-mRNA molecule to generate a mature mRNA that is ready for translation and protects the mRNA from degradation.

5' capping

5' capping is a posttranscriptional modification that adds a unique structure called a 7-methylguanosine cap (m7G) to the 5' end of the mRNA molecule. This cap is important for several reasons: it protects the mRNA from degradation by exonucleases; it assists in nuclear export of the mRNA; and it helps recruit the translation machinery during protein synthesis. Example: All eukaryotic mRNAs undergo 5' capping as part of their normal maturation process.

3' polyadenylation

3' polyadenylation is the process of adding a long chain of adenine nucleotides (a poly(A) tail) to the 3' end of an mRNA molecule. This process is essential for mRNA stability, nuclear export, and translation efficiency. The poly(A) tail is recognized by poly(A) binding proteins, which play important roles in these processes. Example: Most eukaryotic mRNAs, except for histone mRNAs, have a 3' poly(A) tail added to them during posttranscriptional processing.

RNA splicing

RNA splicing is the process of removing non-coding sequences (introns) from the mRNA and joining the coding sequences (exons) together to create a continuous reading frame for protein synthesis. This process is performed by a large protein-RNA complex called the spliceosome, which recognizes specific sequence elements at intron-exon boundaries and catalyses the splicing reaction. Example: The human β-globin pre-mRNA undergoes splicing to remove two introns and generate the mature mRNA that is subsequently translated into one of the protein subunits of hemoglobin.

Alternative splicing

Alternative splicing is a process that allows a single gene to produce multiple mRNA isoforms by combining exons in different ways during the splicing process. This greatly increases the proteomic complexity of eukaryotes by enabling the production of different protein isoforms from a single gene. Example: The human gene encoding the protein tropomyosin (TPM) undergoes alternative splicing to produce multiple mRNA isoforms, which are translated into distinct protein isoforms that have different roles in muscle contraction and other cellular processes.

Key concepts

5' Capping

One of the first steps in posttranscriptional modification is the addition of the 5' cap to the nascent mRNA transcript. This cap is composed of a 7-methylguanosine molecule attached via a triphosphate bridge to the 5' end of the mRNA. The process of capping occurs shortly after transcription initiation and plays several crucial roles:

• Protection: The 5' cap protects the mRNA from degradation by enzymatic activity, specifically by exonucleases that target the end of RNA molecules.
• Nuclear Export: The cap structure is recognized by a set of proteins that assist in transporting the mature mRNA out of the nucleus into the cytoplasm, where translation occurs.
• Translation Initiation: During protein synthesis, the 5' cap is essential for the ribosome to recognize the mRNA and begin translating it into a protein.

All mRNA molecules in eukaryotic cells, apart from the exceptions like rRNA and tRNA, undergo this 5' capping process as they mature.

3' Polyadenylation

Another key posttranscriptional modification is 3' polyadenylation. This involves adding a tail of adenine nucleotides to the 3' end of the mRNA transcript, known as the poly(A) tail. This modification is crucial for:

• Maturation: Stabilizing the mRNA molecule and preventing its degradation by cellular enzymes.
• Nuclear Export: Similar to the 5' cap, the poly(A) tail aids in the transport of the mRNA from the nucleus to the cytoplasm.
• Translation Efficiency: Enhances the translation of mRNA by facilitating its recognition and engagement by the ribosome.

Most eukaryotic mRNAs receive a poly(A) tail during posttranscriptional processing, with histone mRNAs being a notable exception.

RNA Splicing

RNA splicing is an intricate process where non-coding regions called introns are removed from the pre-mRNA, leaving only the coding regions known as exons. This process is carried out by a dynamic complex known as the spliceosome, a molecular machine composed of proteins and small nuclear RNAs (snRNAs). Splicing ensures that:

• The correct coding sequences are joined together to form a continuous open reading frame for translation.
• Potentially deleterious sequences or non-coding RNA are removed prior to mRNA translation.

A classic example is the human β-globin gene, which has introns that need to be spliced out before producing the functional beta globin protein, an essential component of hemoglobin.

Alternative Splicing

Alternative splicing is a sophisticated mechanism that allows a single gene to yield multiple protein variants. This is achieved by selectively including or excluding certain exons during the splicing process, which can generate different mRNA isoforms from the same pre-mRNA. The benefits are:

• Increased Proteomic Diversity: From a single gene, multiple protein forms can be produced, each potentially having distinct functions or regulatory properties in the cell.
• Tissue-Specific Expression: Alternative splicing can lead to the production of proteins that are specific to certain tissues, enhancing organismal complexity and adaptability.

The TPM gene, encoding tropomyosin, exemplifies alternative splicing, allowing different proteins to be expressed in muscle versus non-muscle cells, adapting functionality to the specific protein needs of each tissue.