Fip1 is a multivalent interaction scaffold for processing factors in human mRNA 3' end biogenesis
- University of Zurich, Switzerland
Abstract
3' end formation of most eukaryotic mRNAs is dependent on the assembly of a ~1.5 megadalton multiprotein complex, that catalyzes the coupled reaction of pre-mRNA cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) constitutes the core of the 3' end processing machinery onto which the remaining factors, including cleavage stimulation factor (CstF) and poly(A) polymerase (PAP), assemble. These interactions are mediated by Fip1, a CPSF subunit characterized by high degree of intrinsic disorder. Here, we report two crystal structures revealing the interactions of human Fip1 (hFip1) with CPSF30 and CstF77. We demonstrate that CPSF contains two copies of hFip1, each binding to the zinc finger (ZF) domains 4 and 5 of CPSF30. Using polyadenylation assays we show that the two hFip1 copies are functionally redundant in recruiting one copy of PAP, thereby increasing the processivity of RNA polyadenylation. We further show that the interaction between hFip1 and CstF77 is mediated via a short motif in the N-terminal 'acidic' region of hFip1. In turn, CstF77 competitively inhibits CPSF-dependent PAP recruitment and 3' polyadenylation. Taken together, these results provide a structural basis for the multivalent scaffolding and regulatory functions of hFip1 in 3' end processing.
Data availability
X-ray diffraction data (atomic coordinates and structure factors) have been submitted to the PDB and will be released upon publication.
-
Crystal structure of human CPSF30 in complex with hFip1Protein Data Bank ID: 7ZYH.
-
Crystal structure of human CstF77 in complex with hFip1Protein Data Bank ID: 7ZY4.
-
Cryo-EM structure of human CPSF160-WDR33-CPSF30-PAS RNA-CstF77 complexPotein Data Bank ID: 6URO.
-
Cryo-EM structure of human CPSF160-WDR33-CPSF30-PAS RNA-CstF77 complexElectron Microscopy Data Bank ID: EMD-20861.
Article and author information
Author details
Funding
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (NCCR RNA and Disease)
- Martin Jinek
Boehringer Ingelheim Fonds
- Lena Maria Muckenfuss
Howard Hughes Medical Institute (55008735)
- Martin Jinek
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Muckenfuss et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,249
- views
-
- 288
- downloads
-
- 6
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Fip1 is a multivalent interaction scaffold for processing factors in human mRNA 3' end biogenesis
eLife 11:e80332.
https://doi.org/10.7554/eLife.80332
Further reading
-
- Chromosomes and Gene Expression
- Genetics and Genomics
Models of nuclear genome organization often propose a binary division into active versus inactive compartments yet typically overlook nuclear bodies. Here, we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Although gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.
-
- Chromosomes and Gene Expression
O-GlcNAcylation is the reversible post-translational addition of β-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. It plays an important role in several cellular processes through the modification of thousands of protein substrates. O-GlcNAcylation in humans is mediated by a single essential enzyme, O-GlcNAc transferase (OGT). OGT, together with the sole O-GlcNAcase OGA, form an intricate feedback loop to maintain O-GlcNAc homeostasis in response to changes in cellular O-GlcNAc using a dynamic mechanism involving nuclear retention of its fourth intron. However, the molecular mechanism of this dynamic regulation remains unclear. Using an O-GlcNAc responsive GFP reporter cell line, we identify SFSWAP, a poorly characterized splicing factor, as a trans-acting factor regulating OGT intron detention. We show that SFSWAP is a global regulator of retained intron splicing and exon skipping that primarily acts as a negative regulator of splicing. In contrast, knockdown of SFSWAP leads to reduced inclusion of a ‘decoy exon’ present in the OGT retained intron which may mediate its role in OGT intron detention. Global analysis of decoy exon inclusion in SFSWAP and UPF1 double knockdown cells indicate altered patterns of decoy exon usage. Together, these data indicate a role for SFSWAP as a global negative regulator of pre-mRNA splicing and positive regulator of intron retention.
