Processing of Viral pre-mRNA

Introduction

  • Instructor Information

    • Name: Dr. Fred Krebs, Ph.D.
    • Position: Associate Professor in the Department of Microbiology & Immunology at Drexel University College of Medicine.
    • Contact: Email - fck23@drexel.edu.
    • Faculty profile available through the university's website.

  • Presentation Overview

    • Focus: Processing of viral pre-mRNA.
    • Required actions for completing the topic:
    • Read each section carefully and take notes.
    • Watch available videos and audio recordings.
    • Complete assessment questions labeled "Check Your Understanding."
    • Post questions or comments on the discussion forum.

  • Content Structure

    • Four sections:
    1. Addition of 5’ Caps and 3’ Poly(A) Tails
    2. Regulated Splicing, Polyadenylation, and Nuclear Export of mRNAs
    3. Regulation of Expression through RNA Editing, Turnover, and Silencing
    4. Processing of Viral Pre-mRNA

Addition of 5’ Caps and 3’ Poly(A) Tails

Overview of mRNA Processing

  • General Understanding
    • Viruses (RNA and DNA) regulate transcription of mRNAs encoding viral proteins using cellular transcription machinery.
    • mRNAs require modifications before becoming mature mRNAs capable of cytoplasmic transport for translation.
    • Factors affecting gene product expression include mRNA stability and translation rate.
    • Viruses adapt these cellular mechanisms to promote replication tailored to their specific characteristics, such as genome type (DNA or RNA), size, and replication site (nucleus vs. cytoplasm).

Key Modifications of mRNA

  • The processing events include:
    • Adding a cap structure to the 5’ end of the RNA transcript.
    • Adding a poly(A) tail to the 3’ end.
    • Splicing to remove introns.
    • Occasional sequence editing.
    • RNA transport to the cytoplasm.

5’ Cap Structure

  • Definition:

    • A methylated guanosine (G) added to the 5’ end of the transcript in a 5’ to 5’ linkage, contrasting with the standard 5’ to 3’ linkages of ribonucleotides.
    • Methyl groups may be added to the ribose sugar of the first and possibly second transcribed ribonucleotides.

  • Functions of the 5’ Cap:

    1. Recognition by Translation Machinery: Tags Pol II transcripts for translation recognition and initiation.
    2. Protection from Degradation: Shields mRNA from RNases (5’ to 3’ exonucleases), crucial for mRNA stability and translation efficacy.
    3. Recognition as 'Self' Transcript: Distinguishes cellular mRNA from foreign RNA; unmodified 5' ends alert cells to potential infection.

  • Capping Mechanism:

    • Co-transcriptionally added: At about 20-30 nucleotides of nascent transcript.
    • Enzymatic Steps:
    1. Removal of a phosphate from the 5’ terminal nucleotide by 5’ triphosphatase.
    2. Addition of a GMP residue by guanylyl transferase.
    3. Methylation at the 7-position of the cap by guanine-7-methyltransferase.
    4. Additional methylation by 2’-O-methyltransferase on ribose sugars of the first and second nucleotide bases.

Viral Capping Mechanisms

  • Viral Strategies for Capping:
    • Cellular Enzymes: Some viruses use cellular capping enzymes and transcription machinery (Pol II).
    • Viral Enzymes: Viruses may also possess their own enzymes for RNA capping.
    • Cap Snatching: Some viruses cleave caps from cellular RNAs to cap their own transcripts, exemplified by negative-stranded RNA viruses like influenza.

3’ Polyadenylation

  • Purpose of Poly(A) Tail:

    • Defines the 3’ terminus of the transcript (up to 200 adenine residues).
    • Protects from degradation, stabilizes mRNA, and facilitates efficient translation initiation.

  • Poly(A) Addition Process:

    • Requires binding of protein factors to specific RNA sequences (e.g., AAUAAA) upstream of the addition site.
    • Involves enzymes such as CPSF (Cleavage and Polyadenylation Specificity Factor) and CSTF (Cleavage Stimulatory Factor) that recruit poly(A) polymerase for tail addition.
    • The process includes:
    1. Cleavage at upstream sequence.
    2. Addition of A residues by poly(A) polymerase.
    3. Binding of Poly(A) Binding Protein (PABP) to enhance processivity.

  • Viral Polyadenylation Techniques:

    • Some RNA viruses utilize reiterative copying of U stretches in their genome.
    • DNA viruses like poxviruses encode their own proteins for polyadenylation in the cytoplasm, utilizing unique mechanisms.

Summary of Key Points

  • Both cellular and viral pre-mRNAs undergo co-transcriptional modifications marking them for translation and protecting against degradation.
  • The addition of caps and poly(A) tails is crucial for mRNA stability and recognition.
  • Viral adaptations often modify these processes to suit replication needs.