Gene Expression Overview

Gene expression in eukaryotic cells consists of transcription, processing, and translation.

Transcription

Location: Occurs in the nucleus of the cell.Molecular Components:

• DNA: Holds the genetic information.

• RNA: Synthesized from the DNA template.

• Types of RNA:

  • Messenger RNA (mRNA): Carries the genetic code from DNA to ribosomes for protein synthesis.

  • Noncoding RNA: Includes nuclear noncoding RNA such as Gomafu, Xist, NEAT1, and NEAT2, which have regulatory roles, including gene expression regulation and chromatin remodeling.

Eukaryotic mRNA Processing Overview

Eukaryotic primary mRNA transcripts undergo extensive enzymatic processing.

Purpose:

• Necessary for exporting mRNA from the nucleus and initiating protein synthesis.

• Ensures the integrity and stability of the mRNA molecule for translation.

Objectives of mRNA Processing:

• Understand its biological significance.

• Learn the molecular mechanisms involved.

• Recognize the role of the C-terminal domain of RNA polymerase II in coordinating mRNA processing, facilitating the recruitment of processing factors during transcription.

Steps in Pre-mRNA Processing

1. Transcription Initiation: RNA polymerase II transcribes the primary mRNA from the DNA template.

2. 5' Capping:

   • A 7-methylguanosine cap is added to the 5' end after the first 20-30 nucleotides are transcribed.

   • Functions:

     • Stabilizes the mRNA molecule against degradation.

     • Required for mRNA export from the nucleus.

     • Recognized by translation initiation factors, aligning mRNA on the ribosome during translation initiation.

3. Intron Splicing:

   • Eukaryotic pre-mRNAs often contain non-coding introns that are removed during splicing by the spliceosome, a complex of small nuclear RNAs (snRNAs) and proteins.

   • The spliceosome recognizes conserved sequence elements in pre-mRNA and catalyzes the removal of introns through a series of transesterification reactions.

Why Splice mRNAs?The exon-intron arrangement facilitates the evolution of new protein types and allows for alternative splicing, which increases the coding capacity of the genome, creating diverse protein isoforms from a single gene.

Alternative Splicing

General Concepts:

• Produces various protein isoforms by skipping certain exons, ultimately affecting protein function and regulatory mechanisms.

• Example: Tropomyosin has multiple isoforms which interact with the cytoskeletal structure, showcasing how alternative splicing can yield proteins with different functional capacities.

Significance in Humans:

• The human genome has approximately 20,000 genes, with around 90% capable of undergoing alternative splicing.

• This mechanism enables the estimated expression of roughly 100,000 distinct proteins, significantly enhancing functional diversity in cells.

Mechanism of Pre-mRNA Splicing

Splicing Reaction Steps:

1. U1 & U2 snRNPs Binding: Identify and bind the 5' and 3' splice sites and branch point within the pre-mRNA.

2. Catalytic Spliceosome Formation: A complex is formed using multiple snRNPs, facilitating the formation of a lariat structure.

3. Cleavage & Joining of Exons: Following the removal of introns, the remaining exons are ligated together to form a continuous coding sequence.

Polyadenylation

Addition of a poly(A) tail:

• About 200 adenines are added to the 3' end of mRNA, occurring after cleavage of the pre-mRNA.

• Catalyzed by poly A polymerase, this step is crucial for the mRNA's stability, facilitating export from the nucleus and initiating translation.

Polyadenylation Signals:

• Key sequence elements:

  • AAUAAA: Poly(A) signal sequence, which directs the polyadenylation machinery.

  • G-U rich element: Assists in the cleavage step.

  • Upstream element: Enhances polyadenylation efficiency by promoting binding of polyadenylation factors.

Key Takeaways

Eukaryotic mRNAs undergo extensive processing, including capping, splicing, and polyadenylation. These modifications are crucial for mRNA stability, nuclear export, and successful translation initiation, reflecting the complexity of gene expression regulation in eukaryotic cells.