RBBP6 activates the pre-mRNA 3′ end processing machinery in humans

  1. Vytaute Boreikaite
  2. Thomas S. Elliott
  3. Jason W. Chin
  4. Lori A. Passmore

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Abstract

3′ end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3′ end of the mature transcript, which is important for mRNA localization, translation, and stability. Cleavage must therefore be tightly regulated. Here, we reconstituted specific and efficient 3′ endonuclease activity of human CPSF with purified proteins. This required the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm), and, importantly, the multidomain protein RBBP6. Unlike its yeast homolog Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to humans. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in mRNA processing.

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  1. Version published to 10.1101/gad.349223.121
  2. ASAPbio crowd review

    This review reflects comments and contributions by Karen Lange, Rachel Lau, Claudia Molina, Mafalda Pimentel, Sree Rama Chaitanya Sridhara and Sagar Varankar. Review synthesized by Sree Rama Chaitanya Sridhara.

    The 3’ end of pre-mRNA molecule in metazoan cells is processed to make functional mRNA molecules. This involves chopping the ends to make free 3’ ends that are polyadenylated (or poly-A) to form mature mRNA molecules with a poly-A tail. The 3’ end processing is important for the stability and transport of mature mRNA molecules, transcription, and translation. 3’ end processing is achieved by a 7-subunit cleavage and polyadenylation specificity factor (CPSF). Despite functional genomics and some in vitro reconstitution experiments, the full protein composition and detailed molecular mechanisms of CPSF are not clear.

    The current study reconstitutes the CPSF complex and the preprint reports that an accessory protein called RBBP6 is required for the activation of 3’end cleavage in an RNA-dependent manner. Altogether, the manuscript and data are very clear, and the claims are supported by appropriate experiments and respective controls. The schematics are extremely helpful for the reader to grasp the interactions of different subunits in this complex and also to understand the experiments. These data (with the "back-to-back" preprint in yeast; Rodrigez-Molina et al. 2021) determine a conserved machinery that efficiently cleaves the 3' end of pre-mRNAs in vitro.

    Results

    Major comments

    1. 'Addition of CStF and CFIIm, either individually or together, failed to activate CPSF. However, addition of RBBP6 activated CPSF in the presence of CStF and CFIIm, promoting efficient cleavage of the pre-mRNA substrate.' – There is a faint 5'-cleavage product in the CPSF/RBBP6 and CPSF/CFIIm/RBBP6 lanes. It would be helpful to provide some comments or discussion about this faint band. (Results – Fig. 1 and Fig. S2)
    2. 'Time-course cleavage assays of SV40 pre-mRNA substrates containing either a canonical PAS (RNAAAUAAA) or a mutant PAS (RNAAACAAA) sequence.' – Were any other mutants (e.g., AAGAAA or AAAAAA) tested as part of the study? (Results)
    3. 'Upon addition of ATP, the 5’ cleavage product band disappears and is replaced by a smear corresponding to polyadenylated 5’ products containing poly(A) tails of variable length.' – The "smear" is not clear. It seems like the addition of ATP made cleavage less efficient because the lower 3'cleavage product appears to be fainter than the no ATP lane as well. It might be important to include some clarification on these points. (Results, Fig. S1D)

    Minor comments

    1. 'likely that the role of RBBP6 is conserved from yeast to human' – It is advisable to include the context under which the role of RBBP6 is conserved (Abstract).
    2. 'in insect cells' – It might be helpful to clarify or discuss the status of post-translational modifications of human proteins expressed in insect cells if any? (Results)
    3. 'Unstructured regions were removed from the CPSF subunits WDR33 and hFip1, and the CFIIm subunit Pcf11 to facilitate purification' – Suggest including some comments on how these deletions don't affect the protein function (Results).
    4. 'We hypothesized that the conserved region of the multi-domain protein RBBP6 (residues 1-335; Supplementary Information) might also be required for endonuclease activation' – Is there a reason for this hypothesis or can a relevant reference be included? (Results)
    5. Kwon et al., 2020 – typo Kwon et al., 2021. (Results)
    6. 'We tested various combinations of 3’-end processing factors in cleavage assays and analyzed the results by denaturing gel electrophoresis of RNA (Figure 1C).' – Is this experiment performed with the deletion constructs mentioned earlier? (Results)
    7. 'Overall, we determined that activation of the CPSF endonuclease requires three additional protein factors: CStF, CFIIm and RBBP6' – Does this mean only in vitro or also in vivo? (Results)
    8. 'Each dot represents a single measurement.' – It is not clear how many measurements were taken on a graph. It seems there are more measurements for certain concentrations compared to others. For example, 0.1uM has four compared to 1 at the penultimate highest concentration. Maybe the number of measurements and biological replicates can be described more clearly in the figure legend. (Results, Fig. 2D)
    9. (Sun et al, 2018) – Although a reference is provided, the PDB ID code in the figure legend might be helpful. (Results, Fig. 4D)
    10. 'The DNA sequences encoding fragments of SV40 pre-mRNA with either wild-type (AAUAAA) or mutant PAS (AACAAA)' – It might be helpful to provide the SV70 pre-mRNA sequence for future reference. (Methods).
  3. Version published to 10.1101/2021.11.02.466915v1 on bioRxiv