Dynamics in Fip1 regulate eukaryotic mRNA 3'-end processing

Cleavage and polyadenylation factor (CPF/CPSF) is a multiprotein complex essential for mRNA 3ʹ-end processing in eukaryotes. It contains an endonuclease that cleaves pre-mRNAs, and a polymerase that adds a poly(A) tail onto the cleaved 3ʹ-end. Several CPF subunits, including Fip1, contain intrinsically-disordered regions (IDRs). IDRs within multiprotein complexes can be flexible, or can become ordered upon interaction with binding partners. Here, we show that yeast Fip1 anchors the poly(A) polymerase Pap1 onto CPF via an interaction with zinc finger 4 of another CPF subunit, Yth1. We also reconstitute a fully recombinant 850-kDa CPF. By incorporating selectively-labelled Fip1 into recombinant CPF, we could study the dynamics of this single protein within the megadalton complex using nuclear magnetic resonance spectroscopy (NMR). This reveals that a Fip1 IDR that connects the Yth1- and Pap1-binding sites remains highly dynamic within CPF. Together, our data suggest that Fip1 dynamics mediate conformational transitions within the 3ʹ-end processing machinery to coordinate cleavage and polyadenylation.

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Dynamics in Fip1 regulate eukaryotic mRNA 3′ end processing

Genes & Development ◽

10.1101/gad.348671.121 ◽

2021 ◽

Author(s):

Ananthanarayanan Kumar ◽

Conny W.H. Yu ◽

Juan B. Rodríguez-Molina ◽

Xiao-Han Li ◽

Stefan M.V. Freund ◽

...

Keyword(s):

Nuclear Magnetic Resonance ◽

Nmr Spectroscopy ◽

Magnetic Resonance ◽

Binding Sites ◽

Multiprotein Complex ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Binding Partners ◽

Cleavage And Polyadenylation ◽

Polyadenylation Factor

Cleavage and polyadenylation factor (CPF/CPSF) is a multiprotein complex essential for mRNA 3′ end processing in eukaryotes. It contains an endonuclease that cleaves pre-mRNAs, and a polymerase that adds a poly(A) tail onto the cleaved 3′ end. Several CPF subunits, including Fip1, contain intrinsically disordered regions (IDRs). IDRs within multiprotein complexes can be flexible, or can become ordered upon interaction with binding partners. Here, we show that yeast Fip1 anchors the poly(A) polymerase Pap1 onto CPF via an interaction with zinc finger 4 of another CPF subunit, Yth1. We also reconstitute a fully recombinant 850-kDa CPF. By incorporating selectively labeled Fip1 into recombinant CPF, we could study the dynamics of Fip1 within the megadalton complex using nuclear magnetic resonance (NMR) spectroscopy. This reveals that a Fip1 IDR that connects the Yth1- and Pap1-binding sites remains highly dynamic within CPF. Together, our data suggest that Fip1 dynamics within the 3′ end processing machinery are required to coordinate cleavage and polyadenylation.

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The Arabidopsis autophagy cargo receptors ATI1 and ATI2 bind to ATG8 via intrinsically disordered regions and are post-translationally modified upon ATG8 interaction

10.1101/406538 ◽

2018 ◽

Author(s):

Ida Marie Zobbe Sjøgaard ◽

Simon Bressendorff ◽

Andreas Prestel ◽

Swathi Kausika ◽

Emilie Oksbjerg ◽

...

Keyword(s):

Molten Globule ◽

Resonance Spectroscopy ◽

Wild Type ◽

Post Translational Modification ◽

Interacting Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Specific Proteins ◽

Modified Forms ◽

Disordered Regions

AbstractSelective autophagy has emerged as an important mechanism by which eukaryotic cells control the abundance of specific proteins. This mechanism relies on cargo recruitment to autophagosomes by receptors that bind to both the ubiquitin-like AUTOPHAGY8 (ATG8) protein through ATG8 interacting motifs (AIMs) and to the cargo to be degraded. In plants, two autophagy cargo receptors, ATG8 Interacting Protein 1 (ATI1) and 2 (ATI2), were identified early on, but their molecular properties remain poorly understood. Here, we show that ATI1 and ATI2 are transmembrane proteins with long N-terminal intrinsically disordered regions (IDRs). The N-terminal IDRs contain the functional AIMs, and we use nuclear magnetic resonance spectroscopy to directly observe the disorder-order transition of the AIM upon ATG8 binding. Our analyses also show that the IDRs of ATI1 and ATI2 are not equivalent, because ATI2 has properties of a fully disordered polypeptide, while ATI1 has properties consistent with a collapsed pre-molten globule-like conformation Interestingly, wild type ATI1 and ATI2 exist as distinct post-translationally modified forms. Specifically, different forms are detectable upon mutation of the AIM, suggesting that interaction of ATI1 and ATI2 to ATG8 is coupled to a change in their post-translational modification.

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The transmembrane autophagy cargo receptors ATI1 and ATI2 interact with ATG8 through intrinsically disordered regions with distinct biophysical properties

Biochemical Journal ◽

10.1042/bcj20180748 ◽

2019 ◽

Vol 476 (3) ◽

pp. 449-465 ◽

Cited By ~ 5

Author(s):

Ida Marie Zobbe Sjøgaard ◽

Simon Bressendorff ◽

Andreas Prestel ◽

Swathi Kausika ◽

Emilie Oksbjerg ◽

...

Keyword(s):

Molten Globule ◽

Resonance Spectroscopy ◽

Amino Acid Residues ◽

In Planta ◽

Interacting Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Specific Proteins ◽

Sizable Fraction ◽

Disordered Regions

Abstract Selective autophagy has emerged as an important mechanism by which eukaryotic cells control the abundance of specific proteins. This mechanism relies on cargo recruitment to autophagosomes by receptors that bind to both the ubiquitin-like AUTOPHAGY8 (ATG8) protein through ATG8-interacting motifs (AIMs) and to the cargo to be degraded. In plants, two autophagy cargo receptors, ATG8-interacting protein 1 (ATI1) and 2 (ATI2), were identified early on, but their molecular properties remain poorly understood. Here, we show that ATI1 and ATI2 are transmembrane proteins with long N-terminal intrinsically disordered regions (IDRs). The N-terminal IDRs contain the functional AIMs, and we use nuclear magnetic resonance spectroscopy to directly observe the disorder-order transition of the AIM upon ATG8 binding. Our analyses also show that the IDRs of ATI1 and ATI2 are not equivalent, because ATI2 has properties of a fully disordered polypeptide, while ATI1 has properties more consistent with a collapsed pre-molten globule-like conformation, possibly as a consequence of a higher content of π-orbital-containing amino acid residues. Finally, we show that a sizable fraction of ATI2, but not ATI1, is phosphorylated in planta.

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Intrinsic disorder in proteins: a challenge for (un)structural biology met by ion mobility–mass spectrometry

Biochemical Society Transactions ◽

10.1042/bst20120125 ◽

2012 ◽

Vol 40 (5) ◽

pp. 1021-1026 ◽

Cited By ~ 30

Author(s):

Ewa Jurneczko ◽

Faye Cruickshank ◽

Massimiliano Porrini ◽

Penka Nikolova ◽

Iain D.G. Campuzano ◽

...

Keyword(s):

Ion Mobility ◽

Intrinsically Disordered Proteins ◽

Three Dimensional ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

New Methods ◽

Binding Partners ◽

Nanoelectrospray Ionization ◽

And Function

The link between structure and function of a given protein is a principal tenet of biology. The established approach to understand the function of a protein is to ‘solve’ its structure and subsequently investigate interactions between the protein and its binding partners. However, structure determination via crystallography or NMR is challenging for proteins where localized regions or even their entire structure fail to fold into a three-dimensional form. These so called IDPs (intrinsically disordered proteins) or intrinsically disordered regions constitute up to 40% of all expressed proteins, and a much higher percentage in proteins involved in the proliferation of cancer. For these proteins, there is a need to develop new methods for structural characterization which exploit their biophysical properties. IM (ion mobility)–MS is uniquely able to examine both absolute conformation(s), populations of conformation and also conformational change, and is therefore highly applicable to the study of IDPs. The present article details the technique of IM–MS and illustrates its use in assessing the relative disorder of the wild-type p53 DNA-core-binding domain of cellular tumour antigen p53. The IM data were acquired on a Waters Synapt HDMS instrument following nESI (nanoelectrospray ionization) from ‘native’ and low-pH solution conditions.

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