Identification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins

Poly(A) Binding Proteins (PABPs) are major eukaryotic RNA-binding proteins (RBPs) with multiple roles associated with mRNA stability and translation and characterized mainly from multicellular organisms and yeasts. A variable number of PABP homologues are seen in different organisms however the biol...

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Autores principales:	Ludmila A. Assis, Moezio V. C. Santos Filho, Joao R. da Cruz Silva, Maria J. R. Bezerra, Irassandra R. P. U. C. de Aquino, Kleison C. Merlo, Fabiola B. Holetz, Christian M. Probst, Antonio M. Rezende, Barbara Papadopoulou, Tamara D. C. da Costa Lima, Osvaldo P. de Melo Neto
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270
Acceso en línea:	https://doaj.org/article/e31320c7c7a043c5b57cb564871be3f9
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id	oai:doaj.org-article:e31320c7c7a043c5b57cb564871be3f9
record_format	dspace
institution	DOAJ
collection	DOAJ
language	EN
topic	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270
spellingShingle	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Ludmila A. Assis Moezio V. C. Santos Filho Joao R. da Cruz Silva Maria J. R. Bezerra Irassandra R. P. U. C. de Aquino Kleison C. Merlo Fabiola B. Holetz Christian M. Probst Antonio M. Rezende Barbara Papadopoulou Tamara D. C. da Costa Lima Osvaldo P. de Melo Neto Identification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins
description	Poly(A) Binding Proteins (PABPs) are major eukaryotic RNA-binding proteins (RBPs) with multiple roles associated with mRNA stability and translation and characterized mainly from multicellular organisms and yeasts. A variable number of PABP homologues are seen in different organisms however the biological reasons for multiple PABPs are generally not well understood. In the unicellular Leishmania, dependent on post-transcriptional mechanisms for the control of its gene expression, three distinct PABPs are found, with yet undefined functional distinctions. Here, using RNA-immunoprecipitation sequencing analysis we show that the Leishmania PABP1 preferentially associates with mRNAs encoding ribosomal proteins, while PABP2 and PABP3 bind to an overlapping set of mRNAs distinct to those enriched in PABP1. Immunoprecipitation studies combined to mass-spectrometry analysis identified RBPs differentially associated with PABP1 or PABP2, including RBP23 and DRBD2, respectively, that were investigated further. Both RBP23 and DRBD2 bind directly to the three PABPs in vitro, but reciprocal experiments confirmed preferential co-immunoprecipitation of PABP1, as well as the EIF4E4/EIF4G3 based translation initiation complex, with RBP23. Other RBP23 binding partners also imply a direct role in translation. DRBD2, in contrast, co-immunoprecipitated with PABP2, PABP3 and with RBPs unrelated to translation. Over 90% of the RBP23-bound mRNAs code for ribosomal proteins, mainly absent from the transcripts co-precipitated with DRBD2. These experiments suggest a novel and specific route for translation of the ribosomal protein mRNAs, mediated by RBP23, PABP1 and the associated EIF4E4/EIF4G3 complex. They also highlight the unique roles that different PABP homologues may have in eukaryotic cells associated with mRNA translation. Author summary Flagellated protozoa belonging to the Leishmania genus are the causative agents of the Leishmaniasis, neglected tropical diseases with a worldwide distribution. These parasites are well known for unique molecular mechanisms which include a lack of regulation of mRNA synthesis, generally the major stage for the control of gene expression in eukaryotic cells. Regulation of mRNA translation is therefore critical for survival and adaptation during a life cycle involving multiple life forms. Such regulation is likely dependent on RNA binding proteins (RBPs), such as the Poly-A Binding Protein (PABP) that binds to the 3’ ends of the mRNAs. Three PABPs are found in Leishmania, more than in other unicellular organisms, with presumably distinct but undefined roles. Here we identify different mRNAs targets and specific RBPs with whom they interact. We define a specific association between one of the Leishmania PABPs, a single RBP and mRNAs encoding ribosomal proteins, an important subset of cellular mRNAs. Our results shed new light on the interactions required for the translation of these mRNAs, highlighting the relevance of regulating their translation in different organisms and the convergence of regulatory mechanisms acting through their 5’ and 3’ ends. The data also expands on the known PABP roles.
format	article
author	Ludmila A. Assis Moezio V. C. Santos Filho Joao R. da Cruz Silva Maria J. R. Bezerra Irassandra R. P. U. C. de Aquino Kleison C. Merlo Fabiola B. Holetz Christian M. Probst Antonio M. Rezende Barbara Papadopoulou Tamara D. C. da Costa Lima Osvaldo P. de Melo Neto
author_facet	Ludmila A. Assis Moezio V. C. Santos Filho Joao R. da Cruz Silva Maria J. R. Bezerra Irassandra R. P. U. C. de Aquino Kleison C. Merlo Fabiola B. Holetz Christian M. Probst Antonio M. Rezende Barbara Papadopoulou Tamara D. C. da Costa Lima Osvaldo P. de Melo Neto
author_sort	Ludmila A. Assis
title	Identification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins
title_short	Identification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins
title_full	Identification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins
title_fullStr	Identification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins
title_full_unstemmed	Identification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins
title_sort	identification of novel proteins and mrnas differentially bound to the leishmania poly(a) binding proteins reveals a direct association between pabp1, the rna-binding protein rbp23 and mrnas encoding ribosomal proteins
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/e31320c7c7a043c5b57cb564871be3f9
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spelling	oai:doaj.org-article:e31320c7c7a043c5b57cb564871be3f92021-11-18T09:11:35ZIdentification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins1935-27271935-2735https://doaj.org/article/e31320c7c7a043c5b57cb564871be3f92021-10-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8575317/?tool=EBIhttps://doaj.org/toc/1935-2727https://doaj.org/toc/1935-2735Poly(A) Binding Proteins (PABPs) are major eukaryotic RNA-binding proteins (RBPs) with multiple roles associated with mRNA stability and translation and characterized mainly from multicellular organisms and yeasts. A variable number of PABP homologues are seen in different organisms however the biological reasons for multiple PABPs are generally not well understood. In the unicellular Leishmania, dependent on post-transcriptional mechanisms for the control of its gene expression, three distinct PABPs are found, with yet undefined functional distinctions. Here, using RNA-immunoprecipitation sequencing analysis we show that the Leishmania PABP1 preferentially associates with mRNAs encoding ribosomal proteins, while PABP2 and PABP3 bind to an overlapping set of mRNAs distinct to those enriched in PABP1. Immunoprecipitation studies combined to mass-spectrometry analysis identified RBPs differentially associated with PABP1 or PABP2, including RBP23 and DRBD2, respectively, that were investigated further. Both RBP23 and DRBD2 bind directly to the three PABPs in vitro, but reciprocal experiments confirmed preferential co-immunoprecipitation of PABP1, as well as the EIF4E4/EIF4G3 based translation initiation complex, with RBP23. Other RBP23 binding partners also imply a direct role in translation. DRBD2, in contrast, co-immunoprecipitated with PABP2, PABP3 and with RBPs unrelated to translation. Over 90% of the RBP23-bound mRNAs code for ribosomal proteins, mainly absent from the transcripts co-precipitated with DRBD2. These experiments suggest a novel and specific route for translation of the ribosomal protein mRNAs, mediated by RBP23, PABP1 and the associated EIF4E4/EIF4G3 complex. They also highlight the unique roles that different PABP homologues may have in eukaryotic cells associated with mRNA translation. Author summary Flagellated protozoa belonging to the Leishmania genus are the causative agents of the Leishmaniasis, neglected tropical diseases with a worldwide distribution. These parasites are well known for unique molecular mechanisms which include a lack of regulation of mRNA synthesis, generally the major stage for the control of gene expression in eukaryotic cells. Regulation of mRNA translation is therefore critical for survival and adaptation during a life cycle involving multiple life forms. Such regulation is likely dependent on RNA binding proteins (RBPs), such as the Poly-A Binding Protein (PABP) that binds to the 3’ ends of the mRNAs. Three PABPs are found in Leishmania, more than in other unicellular organisms, with presumably distinct but undefined roles. Here we identify different mRNAs targets and specific RBPs with whom they interact. We define a specific association between one of the Leishmania PABPs, a single RBP and mRNAs encoding ribosomal proteins, an important subset of cellular mRNAs. Our results shed new light on the interactions required for the translation of these mRNAs, highlighting the relevance of regulating their translation in different organisms and the convergence of regulatory mechanisms acting through their 5’ and 3’ ends. The data also expands on the known PABP roles.Ludmila A. AssisMoezio V. C. Santos FilhoJoao R. da Cruz SilvaMaria J. R. BezerraIrassandra R. P. U. C. de AquinoKleison C. MerloFabiola B. HoletzChristian M. ProbstAntonio M. RezendeBarbara PapadopoulouTamara D. C. da Costa LimaOsvaldo P. de Melo NetoPublic Library of Science (PLoS)articleArctic medicine. Tropical medicineRC955-962Public aspects of medicineRA1-1270ENPLoS Neglected Tropical Diseases, Vol 15, Iss 10 (2021)

Identification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins

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