RNA Metabolism: RNA Processing Notes

Overview of Eukaryotic RNA Processing

  • RNA polymerase II coordinates the processing of precursor mRNA.
  • Key steps:
    • Capping
    • Polyadenylation
    • Transcription termination
  • Ribozymes mediate RNA cleavage and splicing reactions.
  • Structure and function of spliceosomes are critical for splicing.
  • A single gene can produce multiple different mRNA transcripts.

RNA Transcripts and Splicing

  • Co-transcriptional splicing: Splicing occurs before transcription is complete.
  • Example: Human dystrophin gene has 79 exons, spans ~2,400 kb, takes ~16 hours to transcribe at ~40 nucleotides/sec.
    • Introns: non-coding sequences that must be removed.
    • Exons: coding sequences that remain.
  • The 5′ end of mRNA is capped with 7-methylguanylate; protects RNA from exonuclease.
  • The 3′ end is typically extended by a poly(A) tail.

Phosphorylation States of RNA Polymerase II

  • Phosphorylation states of the C-terminal domain (CTD) dictate the activity of RNA polymerase II during transcription:
    • Phosphorylation levels vary during stages:
    • PIC assembly
    • Promoter clearance and pausing for capping
    • Productive elongation
  • Different phosphorylation states recruit RNA processing factors.

RNA Capping

  • The capping process involves:
    1. Hydrolysis of phosphate on the 5′ nucleotide (by RNA Triphosphatase).
    2. Addition of GMP (by RNA guanylyltransferase).
    3. Methylation of guanine to form the cap (by guanine methyltransferase).

Polyadenylation

  • A poly(A) tail is added to the 3′ end of transcripts in two steps:
    1. Cleavage occurs downstream of the AAUAAA sequence (3’ UTR).
    2. A's (80-250) are added to form the poly(A) tail.
  • The process requires:
    • CPSF (cleavage/polyadenylation specificity factor)
    • CstF (cleavage stimulatory factor)
    • PAP (poly(A) polymerase)

Splicing Mechanisms

  • Intron Splicing: Four classes:
    1. Group I and II: Self-splicing, do not require specific factors.
    • Group I uses guanine nucleoside cofactor.
    • Group II uses an A residue within the intron forming a lariat structure.
    1. Group III: Eukaryotic spliceosomal introns (largest class).
    2. Group IV: Some tRNAs that require ATP and an endonuclease.
  • Ribozymes mediate many RNA processing reactions.
  • The spliceosome consists of ~150 proteins and 5 snRNAs that carry out splicing.

Spliceosome Assembly and Function

  • Spliceosome recognizes splice sites through short consensus sequences surrounding 5′ and 3′ splice sites.
  • Major steps:
    1. U1 binds to the 5′ splice junction.
    2. U2 binds the intron branch point.
    3. U4/U6 and U5 snRNPs join to form the catalytic spliceosome.
  • The excision of the intron occurs via two transesterification reactions, forming a lariat structure.

Regulatory Factors in Splicing

  • Exonic splicing enhancers (ESEs) and SR proteins influence splicing decision.
  • Splicing factors coordinate with the phosphorylated CTD to enhance processing specificity.

Alternative Splicing

  • Alternative splicing allows a single gene to generate multiple mRNA variants, leading to diverse protein products.
  • Regulated by activators and repressors; splicing machinery can recognize different splicing signals based on cellular context.

Pathophysiology Implications of Splicing

  • Splicing defects can lead to diseases by affecting genetic diversity and causing mis-splicing.
  • Example: IRE1α-XBP1s splicing pathway is involved in cancer signaling (UPR - Unfolded Protein Response).

Summary of Key Points

  • All RNA is synthesized via template-dependent copying by RNA polymerase.
  • Eukaryotic pre-mRNA undergoes extensive processing including capping, splicing, and polyadenylation.
  • The spliceosome plays a critical role in recognizing, excising introns, and ligating exons. Alternative splicing allows for versatility in protein synthesis.

Review Questions

  • Discuss the importance of the phosphorylation states of RNA polymerase II CTD in splicing and transcription.
  • Compare the mechanisms of group I and group II introns.
  • Describe the role of snRNPs in the splicing process and their structural importance in the spliceosome.