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The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation.

ABSTRACT: The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA. We identify Brat-binding sites distinct from the Pum consensus motif and show that RNA binding and translational repression by Brat do not require Pum, suggesting so far unrecognized Pum-independent Brat functions. Using various biochemical and biophysical methods, we also demonstrate that the NHL (NCL-1, HT2A, and LIN-41) domain of Brat, a domain previously believed to mediate protein-protein interactions, is a novel, sequence-specific ssRNA-binding domain. The Brat-NHL domain folds into a six-bladed ? propeller, and we identify its positively charged top surface as the RNA-binding site. Brat belongs to the functional diverse TRIM (tripartite motif)-NHL protein family. Using structural homology modeling, we predict that the NHL domains of all TRIM-NHL proteins have the potential to bind RNA, indicating that Brat is part of a conserved family of RNA-binding proteins.

SUBMITTER: Loedige I

PROVIDER: S-EPMC4015489 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation.

Loedige Inga I Stotz Mathias M Qamar Saadia S Kramer Katharina K Hennig Janosch J Schubert Thomas T Löffler Patrick P Längst Gernot G Merkl Rainer R Urlaub Henning H Meister Gunter G

Genes & development 20140401 7

The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA. We identify Brat-binding sites distinct from the Pum consensus motif and show ...[more]

PMID: 24696456

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Project description:The TRIM-NHL family of proteins shares a conserved domain architecture and play crucial roles in stem cell biology, fertility, and development. This review synthesizes new insights that have revolutionized our understanding of the molecular and biological functions of TRIM-NHL proteins. Multiple TRIM-NHLs have been shown to bind specific RNA sequences and structures. X-ray crystal structures of TRIM-NHL proteins in complex with RNA ligands reveal versatile modes of RNA recognition by the NHL domain. Functional and genetic analyses show that TRIM-NHL RNA-binding proteins negatively regulate the protein expression from the target mRNAs that they bind. This repressive activity plays a crucial role in controlling stem cell fate in the developing brain and differentiating germline. To highlight these paradigms, we focus on several of the most-extensively studied TRIM-NHL proteins, specifically Drosophila and vertebrate TRIM71, among others. Brat is essential for development and regulates key target mRNAs to control differentiation of germline and neural stem cells. TRIM71 is also required for development and promotes stem cell proliferation while antagonizing differentiation. Moreover, TRIM71 can be utilized to help reprogram fibroblasts into induced pluripotent stem cells. Recently discovered mutations in TRIM71 cause the neurodevelopmental disease congenital hydrocephalus and emphasize the importance of its RNA-binding function in brain development. Further relevance of TRIM71 to disease pathogenesis comes from evidence linking it to several types of cancer, including liver and testicular cancer. Collectively, these advances demonstrate a primary role for TRIM-NHL proteins in the post-transcriptional regulation of gene expression in crucial biological processes. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Translation Regulation RNA Turnover and Surveillance > Regulation of RNA Stability.

Project description:BackgroundRLSB, an S-1 domain RNA binding protein of Arabidopsis, selectively binds rbcL mRNA and co-localizes with Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within chloroplasts of C3 and C4 plants. Previous studies using both Arabidopsis (C3) and maize (C4) suggest RLSB homologs are post-transcriptional regulators of plastid-encoded rbcL mRNA. While RLSB accumulates in all Arabidopsis leaf chlorenchyma cells, in C4 leaves RLSB-like proteins accumulate only within Rubisco-containing bundle sheath chloroplasts of Kranz-type species, and only within central compartment chloroplasts in the single cell C4 plant Bienertia. Our recent evidence implicates this mRNA binding protein as a primary determinant of rbcL expression, cellular localization/compartmentalization, and photosynthetic function in all multicellular green plants. This study addresses the hypothesis that RLSB is a highly conserved Rubisco regulatory factor that occurs in the chloroplasts all higher plants.ResultsPhylogenetic analysis has identified RLSB orthologs and paralogs in all major plant groups, from ancient liverworts to recent angiosperms. RLSB homologs were also identified in algae of the division Charophyta, a lineage closely related to land plants. RLSB-like sequences were not identified in any other algae, suggesting that it may be specific to the evolutionary line leading to land plants. The RLSB family occurs in single copy across most angiosperms, although a few species with two copies were identified, seemingly randomly distributed throughout the various taxa, although perhaps correlating in some cases with known ancient whole genome duplications. Monocots of the order Poales (Poaceae and Cyperaceae) were found to contain two copies, designated here as RLSB-a and RLSB-b, with only RLSB-a implicated in the regulation of rbcL across the maize developmental gradient. Analysis of microsynteny in angiosperms revealed high levels of conservation across eudicot species and for both paralogs in grasses, highlighting the possible importance of maintaining this gene and its surrounding genomic regions.ConclusionsFindings presented here indicate that the RLSB family originated as a unique gene in land plant evolution, perhaps in the common ancestor of charophytes and higher plants. Purifying selection has maintained this as a highly conserved single- or two-copy gene across most extant species, with several conserved gene duplications. Together with previous findings, this study suggests that RLSB has been sustained as an important regulatory protein throughout the course of land plant evolution. While only RLSB-a has been directly implicated in rbcL regulation in maize, RLSB-b could have an overlapping function in the co-regulation of rbcL, or may have diverged as a regulator of one or more other plastid-encoded mRNAs. This analysis confirms that RLSB is an important and unique photosynthetic regulatory protein that has been continuously expressed in land plants as they emerged and diversified from their ancient common ancestor.

Dataset Information

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation.

Publications

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation.

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