Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Nov 5;10(11):4063-4069.
doi: 10.1534/g3.120.401644.

RPL-4 and RPL-9 ̶Mediated Ribosome Purifications Facilitate the Efficient Analysis of Gene Expression in Caenorhabditis elegans Germ Cells

Marco Nousch  1
Affiliations

RPL-4 and RPL-9 ̶Mediated Ribosome Purifications Facilitate the Efficient Analysis of Gene Expression in Caenorhabditis elegans Germ Cells

Marco Nousch. G3 (Bethesda). .

Abstract

In many organisms, tissue complexity and cellular diversity create a barrier that can hinder our understanding of gene expression programs. To address this problem, methods have been developed that allow for easy isolation of translated mRNAs from genetically defined cell populations. A prominent example is the Translating Ribosome Affinity Purification method also called TRAP. Here, ribosome associated mRNAs are isolated via purification of the ribosomal protein RPL10A/uL1, which is expressed under the control of a tissue specific promoter. Originally developed to investigate gene expression in mouse neurons, it has by now been adopted to many different organisms and tissues. Interestingly, TRAP has never been used successfully to analyze mRNA translation in germ cells. Employing a combination of genetic and biochemical approaches, I assessed several ribosomal proteins for their suitability for TRAP using the Caenorhabditis elegans germline as a target tissue. Surprisingly, I found that RPL10A/uL1 is not the ideal ribosomal component to perform such an analysis in germ cells. Instead other proteins such as RPL4/uL4 or RPL9/eL6 are much better suited for this task. Tagged variants of these proteins are well expressed in germ cells, integrated into translating ribosomes and do not influence germ cell functions. Furthermore, germ cell-specific mRNAs are much more efficiently co-purified with RPL4/uL4 and RPL9/uL6 compared to RPL10A/uL1. This study provides a solid basis upon which future germ cell TRAP experiments can be built, and it highlights the need for rigorous testing when adopting such methods to a new biological system.

Keywords: gene expression; germ cell; ribosomal protein; translation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
A differential expression is detected for tagged ribosomal proteins in germ cells. (A-B) Structure of the yeast 80S ribosome. RPL-4, RPL-9 and RPL-22 are shown in red. The arrow head indicates the position of the tag. Ribosomal RNA is shown in gray, proteins of the small subunit in light blue and proteins of the large subunit in pink. The original PyMOL file for the shown structure was generated by the Ban lab (https://bangroup.ethz.ch/research/nomenclature-of-ribosomal-proteins.html). (C) Top: Shown is the general structure of the expression constructs; Bottom: Genomic structure of rpl-1, rpl-4, rpl-9 and rpl-22. Gray boxes indicate regions that define the different evolutionary conserved RPL protein regions. (D) Western blot analysis of RPL::FLAG expressing strains. Per lane 30 adult hermaphrodite were loaded.
Figure 2
Figure 2
Expression of tagged RPLs in germ cells has no influence on fecundity. The fertility of parental hermaphrodites (n) was analyzed by counting hatched F1 larvae (progeny). (A) At 20°, no significant difference was detected in the number of median offspring generated by wild type or rpl::flag expressing worms. (B) At 25°, all rpl::flag expressing strains produce less progeny compared to wild type. (C) Western blot analysis of rpl::flag expressing strains grown at different temperatures. No obvious differences in expression levels are detected.
Figure 3
Figure 3
Differential integration of tagged ribosomal proteins into translating ribosomes. A polysome analysis was conducted comparing wild type to rpl::flag expressing animals. (A) Absorbance traces at 260 nm of polysomal gradients from wild type, rpl-1::flag, rpl-4::flag, rpl-9::flag and rpl-22::flag worms. Similar amounts of cellular proteins were loaded onto each gradient. The positions of the major ribosomal complexes are shown on the top. The numbers on the bottom indicate the fractions that were collected. (B) Western blot analysis of the gradient fractions. RPS-5 distribution was similar in all gradients. The image shown was generated from the rpl-22::flag gradient.
Figure 4
Figure 4
Translationally active mRNAs can be efficiently co-purified with RPL-4::FLAG and RPL-9::FLAG. (A) Western blot analysis of copurified RPL::FLAG proteins from adult worms. Equal amount of extract and anti-FLAG beads were used in the different immunopurifications. Input and bead material were analyzed for the indicated proteins. The asterisk in the FLAG blot markes an IgG band. (B) RNA was isolated from input and bead material and analyzed on a denaturing RNA gel stained with ethidium bromide for the presence of the 18S and 26S rRNA. Equal fractions of bead material were loaded for each immunoprecipitation. The signal in the input represents 1% of the amount of extract that was incubated with the bead material. (C) Analysis of mRNA enrichments in the different purifications using quantitative real time PCR.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Anger A. M., Armache J. P., Berninghausen O., Habeck M., Subklewe M. et al. , 2013. Structures of the human and Drosophila 80S ribosome. Nature 497: 80–85. 10.1038/nature12104 - DOI - PubMed
    1. Austin J., and Kimble J., 1987. glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans. Cell 51: 589–599. 10.1016/0092-8674(87)90128-0 - DOI - PubMed
    1. Ben-Shem A., Garreau de Loubresse N., Melnikov S., Jenner L., Yusupova G. et al. , 2011. The structure of the eukaryotic ribosome at 3.0 A resolution. Science 334: 1524–1529. 10.1126/science.1212642 - DOI - PubMed
    1. Brenner S., 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71–94. - PMC - PubMed
    1. Doyle J. P., Dougherty J. D., Heiman M., Schmidt E. F., Stevens T. R. et al. , 2008. Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell 135: 749–762. 10.1016/j.cell.2008.10.029 - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources