# Deciphering the RNA-binding protein network during endosomal mRNA transport

This ARC [Annoteted Research Context](https://arc-rdm.org/) contains the original data of the Devan et al. publication. 

## Original Publication
S. K. Devan, S. Shanmugasundaram, K. Müntjes, S.H.J. Smits, F. Altegoer, M. Feldbrügge (2024) Deciphering the RNA-Binding protein network during endosomal mRNA transport. PNAS. 121:e240491121
https://www.pnas.org/doi/10.1073/pnas.2404091121

## Table of Contents

1. [Abstract](## Abstract)
2. [Studies](## Studies) 
3. [Assays](## Assays)
4. [Licence](## License)

## Abstract
Microtubule-dependent endosomal transport is crucial for polar growth, ensuring the precise distribution of cellular cargos such as proteins and mRNAs. However, the molecular mechanism linking mRNAs to the endosomal surface remains poorly understood. Here, we present a structural analysis of the key RNA-binding protein Rrm4 from Ustilago maydis. Our findings reveal a different type of MademoiseLLE domain (MLLE) featuring a seven-helical bundle that provides a distinct binding interface. A comparative analysis with the canonical MademoiseLLE domain of the poly(A)-binding protein Pab1 disclosed unique characteristics of both domains. Deciphering the MLLE binding code enabled prediction and verification of previously unknown Rrm4 interactors containing short linear motifs. Importantly, we demonstrated that the human MLLE domains, such as those of PABPC1 and UBR5, employed a similar principle to distinguish among interaction partners. Thus, our study provides detailed mechanistic insights into how structural variations in the widely distributed MLLE domain facilitate mRNA attachment during endosomal transport.

## Studies

The [Studies (S)](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/studies?ref_type=heads) are named after the names of the chapters in the publication. 

S1_MLLE3 of Rrm4 Constitutes a Different Type of Seven-Helix MLLE Domain  
S2_MLLE3 of Rrm4 Recognizes PAM2-Like Sequences with a Defined Binding Pocket  
S3_Identifying an Essential FxY Core in PAM2-Like Peptides of Upa1  
S4_MLLE3 of Rrm4 Is Necessary and Sufficient for Endosomal Attachment  
S5_Deciphering the Binding Code for MLLE of Pab1 and MLLE3 of Rrm4  
S6_Identification of Unrecognized Rrm4 Interaction Partners  
S7_Human MLLE Domains Differentiate between Binding Partners  

## Assays
The [Assays (A)](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays?ref_type=heads) folder contains the results of the individual experiments. The raw and processed data are stored in the `dataset` folder of the assay and the corresponding protocols are in the `protocol` folder. 

### Chapter 1: MLLE3 of Rrm4 Constitutes a Different Type of Seven-Helix MLLE Domain 

[S1_A1_Protein structure prediction](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S1_A1_Protein%20structure%20prediction?ref_type=heads) Figure 1B, Figure S1F  
[S1_A2_Protein Purification for structural studies](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S1_A2_Protein%20Purification%20for%20structural%20studies?ref_type=heads) Figure S1A  
[S1_A3_Size exclusion chromatography of H-Rrm4-MLLE3](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S1_A3_Size%20exclusion%20chromatography%20of%20H-Rrm4-MLLE3?ref_type=heads) Figure S1B  
[S1_A4_Western blot analysis of GST pull-down experiments with MLLE3 versions](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S1_A4_Western%20blot%20analysis%20of%20GST%20pull-down%20experiments%20with%20MLLE3%20versions?ref_type=heads) Figure 1C, Figure S1C  
[S1_A5_Isothermal Titration Calorimetry_ITC_using Rrm4 MLLE3](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S1_A6_X-ray%20structure%20of%20Rrm4-MLLE3?ref_type=heads) Figure 1D, Figure S1D  
[S1_A6_X-ray structure of Rrm4-MLLE3](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S1_A6_X-ray%20structure%20of%20Rrm4-MLLE3?ref_type=heads) Figure 1B, Figure S1F  
[S1_A7_Multiple sequence alignment of Rrm4-MLLE3 orthologs of representative fungi from Basidiomycota](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S1_A7_Multiple%20sequence%20alignment%20of%20Rrm4-MLLE3%20orthologs%20of%20representative%20fungi%20from%20Basidiomycota?ref_type=heads) Figure S1F  

### Chapter 2: MLLE3 of Rrm4 Recognizes PAM2-Like Sequences with a Defined Binding Pocket
[S2_A1_Structural analysis of MLLE3 of Rrm4](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S2_A1_Structural%20analysis%20of%20MLLE3%20of%20Rrm4?ref_type=heads) Figure 2A, Figure 2B  
[S2_A2_Western blot analysis of GST pull-down experiments with MLLE3 variants](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S2_A2_Western%20blot%20analysis%20of%20GST%20pull-down%20experiments%20with%20MLLE3%20variants?ref_type=heads) Figure S2A  

### Chapter 3: Identifying an Essential FxY Core in PAM2-Like Peptides of Upa1
[S3_A1_Western blot analysis of GST pull-down experiments with GST-MLLE3 and PAM2L variants](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S3_A1_Western%20blot%20analysis%20of%20GST%20pull-down%20experiments%20with%20GST-MLLE3%20and%20PAM2L%20variants?ref_type=heads) Figure S2A-C, Figure S2G  
[S3_A2_Structural comparison of MLLE3 in U_maydis and R_irregularis](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S3_A2_Structural%20comparison%20of%20MLLE3%20in%20U_maydis%20and%20R_irregularis?ref_type=heads) Figure 2B, Figure S2D-F  
[S3_A3_Western blot analyis of Rrm4-MLLE3 from U_maydis and R_irregularis](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S3_A3_Western%20blot%20analyis%20of%20Rrm4-MLLE3%20from%20U_maydis%20and%20R_irregularis?ref_type=heads) Figure S2C, Figure S2G  

### Chapter 4: MLLE3 of Rrm4 Is Necessary and Sufficient for Endosomal Attachment
[S4_A1_Hyphal growth of strains expressing Rrm4-Kat with MLLE3 variants in AB33 background](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S4_A1_Hyphal%20growth%20of%20strains%20expressing%20Rrm4-Kat%20with%20MLLE3%20variants%20in%20AB33%20background?ref_type=heads) Figure 3B-C, Figure S3  
[S4_A2_Quantification of hyphal growth of AB33 derivatives expressing Rrm4-MLLE variants](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S4_A2_Quantification%20of%20hyphal%20growth%20of%20AB33%20derivatives%20expressing%20Rrm4-MLLE%20variants?ref_type=heads) Figure 3C  
[S4_A3_Micrographs and Kymograph measurement of Rrm4-Kat_MLLE3 variants movement in hyphae](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S4_A3_Micrographs%20and%20Kymograph%20measurement%20of%20Rrm4-Kat_MLLE3%20variants%20movement%20in%20hyphae?ref_type=heads) Figure 3D, Figure S3B-C  
[S4_A4_Quantification of processive Rrm4-Kat signals - velocity and distance travelled](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S4_A4_Quantification%20of%20processive%20Rrm4-Kat%20signals%20-%20velocity%20and%20distance%20travelled?ref_type=heads) Figure S3D-I  

### Chapter 5: Deciphering the Binding Code for MLLE of Pab1 and MLLE3 of Rrm4
[S5_A1_Protein structure prediction](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S5_A1_Protein%20structure%20prediction?ref_type=heads) Figure S4A-B  
[S5_A2_Purification of H-Pab1-MLLE for structural studies](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S5_A2_Purification%20of%20H-Pab1-MLLE%20for%20structural%20studies?ref_type=heads) Figure S4A  
[S5_A3_Size exclusion chromotography of H-Pab1-MLLE](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S5_A3_Size%20exclusion%20chromotography%20of%20H-Pab1-MLLE?ref_type=heads) Figure S4B  
[S5_A4_Isothermal Titration calorimetry-ITC with H-Pab1-MLLE](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S5_A4_Isothermal%20Titration%20calorimetry-ITC%20with%20H-Pab1-MLLE?ref_type=heads) Figure S4C  
[S5_A5_X-ray structure analysis of H-Pab1-MLLE](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S5_A5_X-ray%20structure%20analysis%20of%20H-Pab1-MLLE?ref_type=heads) Figure S4D  
[S5_A6_Western blot analysis of GST pull-down experiments using G-Pab1-MLLE and PAM2 variants](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S5_A6_Western%20blot%20analysis%20of%20GST%20pull-down%20experiments%20using%20G-Pab1-MLLE%20and%20PAM2%20variants?ref_type=heads) Figure 4C, Figure 4E  
[S5_A7_Multiple sequence alignment of Pab1-MLLE orthologs](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S5_A7_Multiple%20sequence%20alignment%20of%20Pab1-MLLE%20orthologs?ref_type=heads) Figure 4D, Figure S4E  


### Chapter 6: Identification of Unrecognized Rrm4 Interaction Partners

[S6_A1_Multiple Sequence alignment of vps8 and Taf7 orthologs](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S6_A1_Multiple%20Sequence%20alignment%20of%20vps8%20and%20Taf7%20orthologs?ref_type=heads) Figure 5A, Figure S5A-B  
[S6_A2_Protein structure prediction using Alphafold2](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S6_A2_Protein%20structure%20prediction%20using%20Alphafold2?ref_type=heads) Figure 5B, Figure S5C, Figure S5E  
[S6_A3_Western blot analysis of GST pull-down experiments using predicted PAM2L variants](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S6_A3_Western%20blot%20analysis%20of%20GST%20pull-down%20experiments%20using%20predicted%20PAM2L%20variants?ref_type=heads) Figure 5C,Figure S5D  
[S6_A4_Hyphal growth of strains expressing Rrm4-Kat and Vps8-Gfp variants in AB33 background](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S6_A4_Hyphal%20growth%20of%20strains%20expressing%20Rrm4-Kat%20and%20Vps8-Gfp%20variants%20in%20AB33%20background?ref_type=heads) Figure S6A-C  
[S6_A5_Quantification of hyphal growth of AB33 derivatives expressing Rrm4-Kat and Vps8-Gfp variants](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S6_A5_Quantification%20of%20hyphal%20growth%20of%20AB33%20derivatives%20expressing%20Rrm4-Kat%20and%20Vps8-Gfp%20variants?ref_type=heads) Figure S6 A-B  
[S6_A6_Micrographs and Kymograph measurement of Rrm4-Kat movement in Vps8-Gfp variant hypha](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S6_A6_Micrographs%20and%20Kymograph%20measurement%20of%20Rrm4-Kat%20movement%20in%20Vps8-Gfp%20variant%20hypha?ref_type=heads) Figure 5E, Figure S7D  
[S6_A7_Quantification of processive Rrm4-Kat signals_velocity and distance travelled in vps8-gfp variant hypha](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S6_A7_Quantification%20of%20processive%20Rrm4-Kat%20signals_velocity%20and%20distance%20travelled%20in%20vps8-gfp%20variant%20hypha?ref_type=heads) Figure S7  
[S6_A8_Quantification of Rrm4-Kat signal accumulated at the tip](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S6_A8_Quantification%20of%20Rrm4-Kat%20signal%20accumulated%20at%20the%20tip?ref_type=heads) Figure S6C  

### Chapter 7: Human MLLE Domains Differentiate between Binding Partners

[S7_A1_Multiple sequence alignment of Mkrn1 orthologs](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S7_A1_Multiple%20sequence%20alignment%20of%20Mkrn1%20orthologs?ref_type=heads) Figure 6A-B, Figure S8A  
[S7_A2_Protein structure prediction of MKRN1 using Alphafold2](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S7_A2_Protein%20structure%20prediction%20of%20MKRN1%20using%20Alphafold2?ref_type=heads) Figure 6C  
[S7_A3_Western blot analysis of GST pull-down experiments using human PAM2L variants](https://git.nfdi4plants.org/devan/2024_Devan-Sai_et_al_PNAS/-/tree/main/assays/S7_A3_Western%20blot%20analysis%20of%20GST%20pull-down%20experiments%20using%20human%20PAM2L%20variants?ref_type=heads) Figure 6D, Figure S8B  

## License
Copyright © 2024 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).