Figure 1. MARF1 interacts with the DCP1:DCP2 mRNA decapping complex

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Figure 1. MARF1 interacts with the DCP1:DCP2 mRNA decapping complex Figure 1. MARF1 interacts with the DCP1:DCP2 mRNA decapping complex. (A) Schematic diagram of full-length MARF1. (B) Dot plot depicting high-confidence protein interactions identified by affinity purification of FLAG-MARF1 in HEK293 cells. SAINT analysis of two independent experiments was performed and a subset of high-confident preys is presented in this dot plot. Node color represents the average spectral counts, and the node edge color corresponds to the SAINTexpress Bayesian FDR value (BFDR). (C) Western blot analysis of lysates derived from HEK293 cells expressing either FLAG-BirA* or FLAG-BirA*-MARF1 and probed with anti-FLAG antibody. (D) Immunoprecipitation (IP) of FLAG-BirA* and FLAG-BirA*-MARF1 from benzonase–treated HEK293 cell extracts using anti-FLAG antibody. Immunoprecipitated complexes were separated by SDS-PAGE and probed with antibodies against the indicated proteins. (E) Streptavidin pulldowns of biotinylated proteins from benzonase-treated lysates outlined in (C). Precipitated proteins were subjected to SDS-PAGE and probed with antibodies against the indicated endogenous proteins. Inputs represent 2% of total lysates. From: Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs Nucleic Acids Res. Published online October 26, 2018. doi:10.1093/nar/gky1011 Nucleic Acids Res | © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Figure 2. The DCP1:DCP2 complex interacts with the MARF1 C-terminus Figure 2. The DCP1:DCP2 complex interacts with the MARF1 C-terminus. (A) Schematic diagram of full-length MARF1 and MARF1 fragments used in co-precipitation experiments in B through E. Dashed lines indicated the region required for decapping factor association in (E). (B) Western blot analysis of lysates derived from HeLa cells expressing FLAG-tagged MARF1 fragments outlined in (A). (C–E) Immunoprecipitation (IP) of MARF1 fragments outlined in (A) from benzonase–treated HeLa cell extracts using anti-FLAG antibody. Immunoprecipitated complexes were separated by SDS-PAGE and probed with antibodies against the indicated proteins. Inputs represent 2% of lysates. From: Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs Nucleic Acids Res. Published online October 26, 2018. doi:10.1093/nar/gky1011 Nucleic Acids Res | © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Figure 3. MARF1 NYN domain is required to degrade a target mRNA Figure 3. MARF1 NYN domain is required to degrade a target mRNA. (A) Schematic diagram of the basic Renilla luciferase-encoding mRNA reporter, containing five 19-nt BoxB hairpins, interacting with λN-HA-MARF1. (B) Schematic diagram of full-length MARF1 and MARF1 fragments used in tethering assays. (C) Expression levels of λNHA-tagged fusion proteins, as determined by western analysis using anti-HA and anti-actin antibodies. (D) RL activity detected in extracts from HeLa cells expressing the indicated proteins. Cells were cotransfected with constructs expressing the RL-5BoxB reporter, FL, and indicated fusion proteins. Histograms represented normalized mean values of RL activity and mRNA levels from a minimum of three experiments. RL activity values seen in the presence of λNHA-LacZ were set as 100. (E) Representative Northern blot of RL-5BoxB and FL mRNAs levels. From: Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs Nucleic Acids Res. Published online October 26, 2018. doi:10.1093/nar/gky1011 Nucleic Acids Res | © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Figure 4. Structure of the MARF1 NYN domain Figure 4. Structure of the MARF1 NYN domain. (A) Comparative sequence analysis of the NYN domains of human (Hs), Xenopus tropicalis (Xt), Danio rerio (Dr), Lasius niger (Ha) and Apis mellifera (Am) MARF1 orthologs. The multiple sequence alignment was generated using MAFFT version 7 and formatted using Jalview (49,50). Secondary structure elements with corresponding numbering are indicated above the sequence. Invariant residues are coloured dark blue while conservative substitutions are depicted in shades of light blue. (B) Left, Crystal structure of the MARF1 NYN domain shown in cartoon and surface representations. Right, cartoon representation of the MARF1 NYN domain, with invariant active site residues depicted in stick format. (C) Structural superpositions of the human MARF1 NYN domain with the NYN domain of Bacillus subtilis YacP/Rae1 (PDB 5MQ8), and the PIN domains of human SMG6 (PDB 2HWW) and MCPIP1 (PDB 3V34). The structures were superimposed using the DALI server (51) and are shown in identical orientations. (D) Zoom-in view of the NYN domain ribonuclease active site of MARF1, overlaid with the structures of human SMG6 and MCPIP1 PIN domains. Invariant active site residues are shown in stick format. The bound magnesium ion present in the MCPIP1 structure is depicted as a purple sphere. From: Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs Nucleic Acids Res. Published online October 26, 2018. doi:10.1093/nar/gky1011 Nucleic Acids Res | © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Figure 5. The MARF1 NYN displays endoribonuclease activity in vitro Figure 5. The MARF1 NYN displays endoribonuclease activity in vitro. (A) Upper panel: Schematic representation of the wild-type and mutant (MUT) MARF1 NYN proteins used for in vitro degradation assays. Mutant MARF1 protein contains tandem alanine substitutions for Asp426 and Asp427. Lower panel: Coomassie-stained SDS-PAGE gel of recombinant wild-type (WT) or mutant (MUT) MARF1 NYN proteins. (B) NYN<sup>WT</sup> protein was incubated with a 5′-<sup>32</sup>P-end-labelled U<sub>30</sub> oligonucleotide in the presence or absence of 3 mM Mn<sup>2+</sup> or Mg<sup>2+</sup>. RNA was extracted at defined time points and resolved via denaturing PAGE. RNA signals were visualized by autoradiography. OH: hydrolysis ladder. (C) Time course analysis of NYN<sup>WT</sup> and NYN<sup>MUT</sup> proteins incubated with a 5′-<sup>32</sup>P-end-labelled U<sub>30</sub> oligonucleotide in the presence of Mn<sup>2+</sup>. OH: hydrolysis ladder. (D) NYN<sup>WT</sup> protein was incubated in the presence of Mn<sup>2+</sup> with a 3′-<sup>32</sup>P-end-labelled U<sub>30</sub> oligonucleotide. RNA was extracted at defined time points and resolved via denaturing PAGE. OH: hydrolysis ladder. From: Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs Nucleic Acids Res. Published online October 26, 2018. doi:10.1093/nar/gky1011 Nucleic Acids Res | © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Figure 6. MARF1 NYN displays endonuclease activity in vivo Figure 6. MARF1 NYN displays endonuclease activity in vivo. (A and E) Expression levels of λNHA-tagged fusion proteins, as determined by Western analysis using anti-HA and anti-actin antibodies. (B) RL activity detected in extracts from HeLa cells expressing the indicated proteins. Cells were cotransfected with constructs expressing the RL-5BoxB reporter, FL, and indicated fusion proteins. Histograms represented normalized mean values of RL activity and mRNA levels from a minimum of three experiments. RL activity values seen in the presence of λNHA-LacZ were set as 100. (C) Representative Northern blot of RL-5BoxB and FL mRNAs levels for (B). (D) Schematic diagram of the Renilla luciferase-encoding mRNA reporter, containing five 19-nt BoxB hairpins, a 114 nt internal poly(A), a 40 nt linker and a self-cleaving hammerhead ribozyme (HHR). HHR cleavage site is denoted by an arrow. (F) Cells were cotransfected with constructs expressing the RL-5BoxB-A114-N40-HHR reporter, FL, and indicated fusion proteins. Histograms represented normalized mean values of RL activity and mRNA levels from a minimum of three experiments. RL activity values seen in the presence of λNHA-LacZ were set as 100. (G) Representative Northern blot of RL-5BoxB-A114-N40-HHR and FL mRNAs levels for (F). From: Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs Nucleic Acids Res. Published online October 26, 2018. doi:10.1093/nar/gky1011 Nucleic Acids Res | © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Figure 7. Model of MARF1-mediated mRNA decay Figure 7. Model of MARF1-mediated mRNA decay. MARF1 binds to a target mRNA via its RRMs. The MARF1 NYN endonuclease cleaves the target mRNA, which is subsequently decapped by the DCP1:DCP2 complex and degraded by XRN1 (not shown). Of note, MARF1-binding sites on targeted mRNAs have yet to be experimentally validated. From: Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs Nucleic Acids Res. Published online October 26, 2018. doi:10.1093/nar/gky1011 Nucleic Acids Res | © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.