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Processing of Viral pre-mRNA

Introduction to Processing of Viral pre-mRNA

  • Instructor: Dr. Fred Krebs, Ph.D. (Associate Professor, Department of Microbiology & Immunology, Drexel University College of Medicine)
  • Course Information:
    • MIIM-512: Molecular Pathogenesis I (Viral Pathogenesis)
    • MIIM-540: Viruses and Viral Infections
  • Scope: Focus on processing mechanisms of viral pre-mRNAs, including modification processes, splicing, polyadenylation, nuclear export, and methods of regulation.

Course Completion Instructions

To successfully navigate this topic:

  1. Read through each section thoroughly, taking notes.
  2. Play the available videos and audio segments to supplement your learning.*
  3. Engage with the "Check Your Understanding" questions throughout the presentation for self-assessment. These questions are not graded. *
  4. Post any questions or comments on the Questions-Problems-Suggestions forum.

*Note: Certain multimedia presentations may not include all resources.

Topic Organization

  • 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
  • Focus: How viral and cellular pre-mRNAs are processed, highlighting similarities and differences in mechanisms.

Section 1: Addition of 5’ Caps and 3’ Poly(A) Tails

  • Overview: This part discusses the significance of cap structures and polyadenylation in mature mRNA production from pre-mRNA synthesized by RNA polymerase II.
    • Processing of many RNA viruses is informed by studies on cellular RNA modifications due to shared mechanisms.

5’ Cap Structure

  • Definition: A 5’ cap is a methylated guanosine residue attached to the 5’ end of mRNA via a unique 5’ to 5’ linkage.
    • This differs from standard 5’ to 3’ phosphodiester bonds linking the rest of the nucleotides.
  • Functionality:
    1. Recognition by The Translation Machinery:
    • Marks RNA Pol II transcripts for the translation machinery, initiating translation accurately.
    1. Protection from Degradation:
    • Prevents mRNA degradation from cellular RNases (specifically 5’ to 3’ exonucleases).
    1. Recognition as Self Transcript:
    • Prevents immune recognition of viral transcripts by marking normal cellular mRNAs.

Mechanisms of Capping

  • Caps are Added Co-transcriptionally: Begins when 20-30 nucleotides emerge from RNA Pol II.
  • Enzymatic Steps Involved in Capping:
    1. Removal of 5’ phosphate (5’ triphosphatase).
    2. Addition of Guanosine (by guanyltransferase).
    3. Methylation of Guanine (by guanine-7-methyltransferase).
    • Additional methylation can occur at ribose sugars of the first two transcription nucleotides (2’-O-methyltransferase).

Variability in Viral Capping Mechanisms

  • Viruses’ Use of Cellular Mechanisms:
    • Some viruses utilize cellular enzymes for capping, while others have their own polymerases to direct the process.
    • Cap Snatching: Certain viruses acquire caps from host mRNA transcripts for capping their own viral mRNAs.

Role of the 3’ Polyadenylation of mRNAs

  • Purpose of Poly(A) Tail:
    1. Defining 3’ Terminus of the transcript.
    2. Protection Against Degradation: Enhances mRNA stability and promotes translation.
  • Polyadenylation Process: Involves specific factors binding to sequences adjacent to poly(A) addition sites to ensure correct cleavage and addition of adenine residues.
    • The enzyme involved is poly(A) polymerase, which synthesizes the tail and is supported by a poly(A) binding protein (PABP) during elongation.

Section 2: Regulated Splicing, Polyadenylation, and Nuclear Export of mRNAs

  • Introduction to Regulated Splicing: Emphasizes the significance of splicing in producing mature mRNA from pre-mRNA by excising introns and joining exons.
    • Events of splicing were initially observed in viral systems, hence reflecting parallels with cellular mechanisms.

Adenoviral Major Late Transcript Splicing

  • Identification of Introns: Evidence of introns was obtained through hybridization studies indicating regions of viral RNA not pairing with DNA.
  • Conserved Sequence Elements: 5’ and 3’ intron ends are marked by conserved sequences (GU and AG) which signal splice sites.

Splicing Mechanism Involvement

  • Splicing Involves snRNPs (Small nuclear ribonucleoproteins): Facilitate the recognition and processing of introns. The process entails different phases, including:
    • Breakage of phosphodiester bonds at splice sites
    • Formation of lariat structures

Alternative Splicing Patterns and Regulation

  • Importance of Alternative Splicing: Enables diverse protein production through varying splicing patterns, beneficial for viruses due to limited genomic capacity.
  • Factors Influencing Splice Site Selection: Splice site outcomes are affected by binding of sequence-specific proteins which regulate splice site choices.

Nuclear Export of Mature mRNAs

  • Recognizes that properly processed mRNAs are transported from the nucleus to the cytoplasm, marking steps associated with translation.

Section 3: Regulation of Expression Through RNA Editing, Turnover, and Silencing

  • Definition of RNA Editing: Modification of nucleotide sequences in RNA; can occur during transcription (co-transcriptional) or post-transcriptionally (after production).
    • Co-transcriptional editing example highlighted in Measles virus altering P gene transcripts through slippage.
  • Post-Transcriptional Editing Example: Hepatitis D virus editing results in two forms of delta antigen due to changes made by the enzyme ADAR.

mRNA Stability and Degradation Processes

  • Regulated mRNA Degradation: Critical in controlling gene expression levels in cells; initiated generally by deadenylation, which reduces stability.

Section 4: RNA Interference and MicroRNAs in Viral Infections

  • RNA Interference (RNAi): An antiviral mechanism against viral invaders, mediated by siRNAs derived from long double-stranded RNA.
    • Viruses have developed means to counteract this with proteins that block RNAi activity.
  • MicroRNAs Role: Small regulatory RNAs produced from host’s genomic regions that influence viral infections indirectly.
    • Example: Hepatitis C virus utilizes miR-122 for replication despite conventional miRNA functions being repression of gene expression.

Conclusion

  • Recap of key themes: Processing of viral pre-mRNAs (including capping, polyadenylation, splicing, and strategies for nucleic acid stabilization)
    • Viruses exhibiting varied adaptations of cellular processes for optimization of replication.

Credits

  • Main Text: Principles of Virology, Volume 1, 5th Edition (J. Flint et al.), ASM Press, 2020.
  • Additional Resources With URLs as Evidenced in the Transcript.