Significant roles in RNA-binding for the amino-terminal regions of Drosophila Pumilio and Nanos
Tammy H. Wharton, Mohammad Marhabaie, Robin P. Wharton
Abstract
The Drosophila Pumilio (Pum) and Nanos (Nos) RNA-binding proteins govern abdominal segmentation in the early embryo, as well as a variety of other events during development. They bind together to a compound Nanos Response Element (NRE) present in thousands of maternal mRNAs in the ovary and embryo, including hunchback (hb) mRNA, thereby regulating poly-adenylation, translation, and stability. Many studies support a model in which mRNA recognition and effector recruitment are carried out by distinct regions of each protein. The well-ordered Pum and Nos RNA-binding domains (RBDs) are sufficient to specifically recognize NREs; the larger intrinsically disordered N-terminal regions (NTRs) of each protein have been thought to act by recruiting mRNA regulators.
Introduction
Translational regulation by Pum and Nos plays an important role in Drosophila, particularly at three stages in development of the ovarian germline and the early embryo. First, the initial step in formation of an egg chamber involves the division of a germline stem cell; maintenance of stem cell status requires Pum and Nos activity [1,2]. Second, from late in oogenesis to the onset of zygotic transcription in the embryo, Pum and Nos jointly regulate thousands of mRNAs to sculpt the maternal transcriptome [3]. Third, Pum and Nos jointly regulate maternal hb mRNA in the posterior of the syncytial cleavage stage embryo to allow abdominal segmentation [4,5].
Materials and method
Fly stocks were maintained by standard methods and grown at 25°C. Transgenic lines were constructed by microinjection of w1118 embryos using standard methods. For the experiments in Fig 4B, abdominal segmentation was scored both in otherwise wt flies and in hbFB/ + heterozygotes. Abdominal segmentation is sensitive to the level of maternal hb mRNA, as reflected in the slight difference in the average number of segments in the two backgrounds (S4 Data). We did not score embryos with the zygotic hbFB/ hbFB phenotype, which has confounding posterior defects and can be readily identified by characteristic head and thoracic defects. Although we did not measure expression of the hb reporter bearing tandem wt NREs, it substantially rescues the zygotic hbFB/ hbFB phenotypes and thus is presumably expressed to normal levels. For measurement of transgene-encoded hb reporter mRNA, total RNA was prepared by homogenization of 0-1 hr embryos. cDNA was then synthesized from 250 ng RNA using random hexamers and qPCR performed using SYBR Green and a 7500 Real Time PCR machine (Applied Biosystems).
Results
As described above, Nos and Pum bind jointly to a compound 11 nt RNA sequence consisting of adjacent binding sites for each protein (Fig 1A). In addition to interactions made by each RBD to the RNA, formation of the ternary complex is dependent on interactions between the two RBDs, involving F1367 in Pum and residues in the C-terminal tail of Nos that are deleted in the L7 mutant (Fig 1A) [4,5,24].
Discussion
Previous work on RNA recognition by Pum and Nos has focused on studies of their RBDs. In this report, we show that the NTRs of both proteins play important roles in RNA binding and discuss models for how they may act.
Acknowledgments
We thank Drs. R Arvola, P Herman, M Parthun, and G Singh for comments on the manuscript, Dr. M Campbell for teaching RPW the rudiments of programming in R, Dr. C Croce for sharing equipment, Drs. M Corchado and H Chamberlin for a gift of wt N2 C. elegans, and Dr. M Wickens for sending yeast strain YBZ1. We acknowledge services of the OSUCCC Genomics Shared Resource which is supported by P30CA016058. The anti-RpL3 mouse monoclonal developed by John Warner was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.
Citation: Wharton TH, Marhabaie M, Wharton RP (2025) Significant roles in RNA-binding for the amino-terminal regions of Drosophila Pumilio and Nanos. PLoS Genet 21(3): e1011616. https://doi.org/10.1371/journal.pgen.1011616
Editor: Elizabeth R. Gavis, Princeton University, UNITED STATES OF AMERICA
Received: December 20, 2024; Accepted: February 11, 2025; Published: March 31, 2025
Copyright: © 2025 Wharton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Funding: This research was supported in part by NIGMS R01GM084376 (RPW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
