L15 Ch8 RNA processing part 2

Page 1: Polyadenylation Complex and 3' End Processing

  • Core processing factors involved in 3' end formation:

    • CPSF (cleavage and polyadenylation specificity factor) binds to the polyadenylation signal sequence.

    • CStF (cleavage stimulation factor) binds to a U-rich region located downstream of the signal.

    • CF I and CF II (cleavage factors I and II) participate in cleavage.

    • PAP (polyadenylate polymerase) also binds to the processing complex.

  • Cleavage and polyadenylation steps:

    • The mRNA is cleaved about 15–30 nucleotides downstream of the polyadenylation signal, i.e. at a distance of roughly
      Δ=15−30 nt\Delta = 15{-}30 \ \text{nt} downstream from the AAUAAA motif.

    • After cleavage, PAP synthesizes the poly(A) tail on the newly formed 3' end.

  • Tail elongation and regulation:

    • The synthesis of the poly(A) tail is accelerated by PABII (poly(A) binding protein II), which binds the nascent tail and enhances processivity.

  • Signals and motifs mentioned:

    • Polyadenylation signal: AAUAAAAAUAAA (canonical signal).

    • Downstream binding region: U-rich sequence where CStF binds.

    • Variant signal in some contexts: AATAAAAATAAA (shown as an alternative in figures).

  • Key takeaways:

    • A coordinated 3' end processing complex (CPSF, CStF, CF I/II, PAP) recognizes the signal and downstream region, cleaves the transcript a short distance downstream, and then PAP adds the poly(A) tail, with tail elongation aided by PABPII.

Page 2: Functions of the Poly(A) Tail

  • Main functional roles of the poly(A) tail:

    • Stability: The tail stabilizes the mRNA and protects it from degradation by exonucleases.

    • Nuclear export: Tail presence facilitates export of mRNA from the nucleus to the cytoplasm.

    • Translation initiation: The poly(A) tail supports ribosome attachment and efficient translation; it is involved in promoting translation initiation and sometimes reinitiation.

  • Additional context (implicit in the transcript):

    • The tail coordinates with associated cap- and translation-related factors to optimize mRNA fate from export to translation.

Page 3: Torpedo Termination Model

  • Conceptual overview:

    • The torpedo model explains transcription termination by coupling 3' end processing with 5' to 3' degradation of the downstream RNA fragment.

    • After cleavage at the poly(A) site, a downstream RNA fragment that is uncapped is degraded by a 5'→3' exonuclease, which ultimately leads to RNA polymerase II termination.

  • Key components and symbols (as depicted in the diagram):

    • RNA polymerase II (RNAP II) with its CTD (C-terminal domain) phosphorylated (P) at various residues, coordinating processing events.

    • 5' cap on the mature mRNA (5' Cap).

    • Polyadenylation signal: AAUAAA (canonical); variant noted as AATAAA in some contexts.

    • 3' cleavage occurs downstream of the poly(A) signal, generating a mature mRNA ending with a poly(A) tail.

    • Torpedo RNase (an endonuclease) initiates degradation of the downstream fragment.

    • Rat1/hXrn2 (5'→3' exonuclease) processively degrades the downstream RNA toward RNAP II.

    • The end result is mature mRNA with a 5' cap and a poly(A) tail, and the downstream fragment is degraded.

  • Stepwise process (as per the torpedo model):
    1) Recognition of the polyadenylation signal (AAUAAA) recruits the 3' end processing machinery (CPSF, CStF, CF I/II, PAP, PABPII).
    2) Cleavage occurs approximately downstream of the AAUAAA signal.
    3) PAP adds the poly(A) tail to the exposed 3' end; PABPII binds the growing tail.
    4) The downstream transcript fragment, lacking a cap, is targeted by Torpedo RNase and by Rat1/hXrn2 in the 5'→3' direction.
    5) Degradation of the downstream fragment by the exonuclease promotes disassembly of the transcription complex and termination of RNAP II.
    6) The mature mRNA features a 5' cap and a 3' poly(A) tail; the residual downstream fragment is removed.

  • Implications and considerations:

    • The torpedo mechanism links cleavage/polyadenylation directly to transcription termination via a chasing exonuclease.

    • If Rat1/hXrn2 function is impaired, termination may fail or be inefficient, potentially causing transcriptional read-through.

    • The canonical polyadenylation signal is AAUAAA; variants like AATAAA can also participate in processing under certain contexts.

  • Connections to broader concepts:

    • The CTD of RNAP II serves as a dynamic platform to recruit capping, splicing, and 3' end processing factors, coordinating the maturation of the RNA with transcription.

    • Proper 3' end formation is essential for mRNA stability, export, and controlled gene expression; disruption can have downstream regulatory and cellular consequences.

  • Quick recap of symbols you should remember:

    • RNAP II: RNA polymerase II

    • CTD: C-terminal domain of RNAP II

    • CPSF, CStF, CF I/II: 3' end processing factors

    • PAP: polyadenylate polymerase

    • PABPII: poly(A) binding protein II

    • Torpedo RNase and Rat1/hXrn2: enzymes driving degradation of the downstream transcript

  • Practical example:

    • If Rat1/hXrn2 is inhibited, the torpedo-driven termination may be compromised, leading to read-through transcription and potential interference with downstream genes.

Notes:

  • All explicit sequence signals mentioned: AAUAAA (polyadenylation signal) and AATAAA (variant).

  • The canonical distance for cleavage relative to AAUAAA is approximately 15–30 nucleotides downstream.

  • The poly(A) tail is synthesized by PAP and elongated with the help of PABPII and other tail-binding factors, contributing to mRNA stability and translational efficiency.