Olsen PH et al. (1997) International C. elegans Meeting "THE 22 NUCLEOTIDE lin-4 REGULATORY RNA IS ONE COMPONENT OF A 9.2S MACROMOLECULAR COMPLEX"

Olsen PH, Ambros VR

[International C. elegans Meeting, 1997]

The steady state level of LIN-14 protein decreases at least 10-fold from the early L1 to the L2 stage as a result of a post-transcriptional mechanism that somehow involves the lin-4 regulatory RNA. The rate of lin-14 transcription, the polyA tail length, and the steady state level of lin-14 mRNA are similar in the L1 and the L2. Furthermore, there is no apparent change in the polysome distribution of lin-14 mRNA from the L1 to the L2. These data suggest that the point of action of lin-4 RNA is downstream of translation initiation (WBG v14, #5, p54). We have begun a comprehensive biochemical characterization of lin-4 RNA to further explore the mechanism of the lin-4 RNA dependent downregulation of LIN-14. Structure studies demonstrate that the 22 nt lin-4 RNA is uncapped and is phosphorylated at the 5' end. Sucrose gradient centrifugation indicates that although approximately 10% of the total cellular lin-4 RNA is associated with the polysome fraction, the other 90% is in a macromolecular complex with a sedimentation coefficient of 9.2S. We are determining the constituents of this apparent lin-4S ribonucleoprotein complex.

Jacqueline M. Brooks et al. (2007) International Worm Meeting "Translational repression of pal-1 maternal mRNA in the C. elegans germline."

Craig P. Hunter, Jacqueline M. Brooks

[International Worm Meeting, 2007]

Translational repression of maternal mRNAs is a fundamental mechanism of gene regulation. PAL-1 is a C. elegans caudal homolog and a maternally expressed gene that patterns posterior embryonic development. Maternal pal-1 transcripts are present throughout the germline and embryo, but PAL-1 protein does not accumulate until the four-cell stage of embryogenesis, and then only in the two posterior blastomeres (Hunter and Kenyon, 1996). It has been shown that GLD-1, a member of the evolutionarily conserved STAR/Maxi-KH domain family, acts through a Germline Repression Element (GRE) in the pal-1 3'' UTR to repress pal-1 translation in the distal germline (Mootz et al., 2004). GLD-1 is a global regulator of germline development in C. elegans, and acts in part by repressing the translation of a repertoire of maternal mRNAs. Interestingly the GRE contains predicted miRNA binding sites that may be involved in selecting the pal-1 mRNA for repression. To investigate the mechanism of GLD-1 mediated translational repression we are using RNAi to target select components implicated in both miRNA mediated processes and translational repression in strains expressing reporters regulated by the pal-1 3UTR control element. Polysome analysis suggests that pal-1 translation is inhibited post-initiation on elongating ribosomes. To elucidate the mechanism(s) of this translational repression we are biochemically probing the process of polysome elongation using both the established rabbit reticulocyte lysate system in parallel with analysis of translation in extracts of C. elegans. These complementary systems are providing important tools for biochemical fractionation and analysis of the mechanism(s) of pal-1 translational repression.

Rochester, J.D. et al. (2019) International Worm Meeting "The Vasa DEAD-box helicase GLH-1 promotes differential translation of sperm genes."

Rochester, J.D., Graber, J., Updike, D., Sharp , C.

[International Worm Meeting, 2019]

P granules are a heterogeneous mix of RNA and protein that reside at the nuclear periphery of germ cells.1 Here they receive nascent transcripts as they exit the nucleus.2 Like germ granules in other animals, P granules in C. elegans contain core Vasa DEAD-box helicases called GLHs (germline helicases).3 Vasa proteins have been implicated in both small RNA amplification4 and translational regulation5, but how this conserved multipotency factor works is unclear. GLH-1 and GLH-2 function redundantly and are the only germline helicases in C. elegans that contain all the domains that distinguish Vasa proteins from other DEAD-box helicases. Loss of both glh-1 and glh-2 causes sterility, but glh-1 single mutants are fertile at permissive temperatures, providing a way to compare relatively healthy germlines from both wild-type and glh-1 mutant worms. mRNA-seq was performed, in triplicate, on total and polysome-associated RNA from a wild-type strain and a strain carrying a precision deletion of glh-1. Like previous microarray studies in glh-1 mutants, very few changes in transcript levels were observed. However, results comparing polysome fractions showed a dependence on GLH-1 for the selective translation of hundreds of sperm-enriched transcripts. In contrast, a handful of histone variant transcripts lose association with polysomes in the glh-1 deletion. To validate these observations, translational reporters of histone and sperm genes are being created in wild-type and glh-1 deletion backgrounds and changes in reporter expression area being compared to the spatial expression and abundance of their transcripts using single molecule FISH. These results are providing insight into the male-specific sterility of Vasa/Mvh/DDX4 mutants in mammals and the mechanism of Vasa function across species. 1. Seydoux, G. J. Mol. Biol. 430, 4702-4710 (2018). 2. Sheth, U. et. al. Development 137, 1305-14 (2010). 3. Spike, C. et al. Genetics 178, 1973-87 (2008). 4. Xiol, J. et al. Cell 157, 1698-1711 (2014). 5. Lasko, P. Biochim. Biophys. Acta - Gene Regul. Mech. 1829, 810-816 (2013).

Jacqueline M Brooks et al. (2005) International Worm Meeting "Translational repression of pal-1 maternal mRNA in the C. elegans germline"

Jacqueline M Brooks, Craig P Hunter, Darcy Mootz

[International Worm Meeting, 2005]

PAL-1 is a caudal homolog that patterns posterior embryonic development in C. elegans. pal-1 transcription begins in the maternal germline, but PAL-1 protein does not accumulate until the four-cell stage of embryogenesis, and then only in the two posterior blastomeres (Hunter and Kenyon, 1996). It has been shown that GLD-1, a member of the evolutionarily conserved STAR/Maxi-KH domain family, acts through the Germline Repression Element (GRE) in the 3' UTR to repress pal-1 translation in the distal germline (Mootz et al., 2004). GLD-1 is a global regulator of germline development in C. elegans, and acts in part by repressing the translation of a repertoire of maternal mRNAs. Interestingly the GRE contains predicted miRNA binding sites that may be involved in selecting the pal-1 mRNA for repression. To investigate the mechanism of GLD-1 mediated translational repression we are using RNAi to target select components implicated in miRNA mediated processes and translational repression in strains expressing reporters regulated by the pal-1 3UTR control element. Polysome analysis suggest that pal-1 translation is inhibited on elongating ribosomes. We hope that the results of these experiments will shed light on the mechanism of post-initiation repression of translation.

Jacqueline Brooks et al. (2006) Development & Evolution Meeting "Translational Repression of pal-1 maternal mRNA in the C. elegans germline"

Craig Hunter, Jacqueline Brooks

[Development & Evolution Meeting, 2006]

PAL-1 is a caudal-like homeodomain protein that specifies posterior embryonic development in C. elegans. Transcription of pal-1 begins in the maternal germline, but PAL-1 protein does not accumulate until the four-cell stage of embryogenesis, and then only in the two posterior blastomeres (Hunter and Kenyon, 1996). It has been shown that GLD-1, a member of the evolutionarily conserved STAR/Maxi-KH domain family, acts through the Germline Repression Element (GRE) in the pal-1 3' UTR to repress its translation in the distal germline (Mootz et al., 2004). GLD-1 is a global regulator essential for germline development in C. elegans, and acts in part by repressing the translation of a repertoire of maternal mRNAs. Interestingly the GRE contains predicted miRNA binding sites that may be involved in targeting the pal-1 mRNA for repression. To investigate the mechanism of GLD-1 mediated translational repression we are using RNAi to target select components implicated in translational repression and miRNA mediated processes in strains expressing reporters regulated by the pal-1 3' UTR control element. Preliminary results suggest that the miRNA machinery contributes to pal-1 translational repression and polysome analysis suggest that pal-1 translation is inhibited on elongating ribosomes. We hope that the results of these experiments will shed light on the mechanism of post-initiation repression of translation.

Coskun, P. et al. (2019) International Worm Meeting "3' UTR mediated translational regulation of glp-1 in the germline of Caenorhabditis elegans."

Coskun, P., Ryder, S.

[International Worm Meeting, 2019]

Translational control of maternal mRNAs is a major form of gene regulation during germline development and embryogenesis. In Caenorhabditis elegans, the maternal gene glp-1 encodes a homolog of the Notch transmembrane protein required for cell proliferation in the germline, and cell fate specification in the embryo. The RNA binding proteins POS-1 and GLD-1 directly regulate the translation of GLP-1 protein by binding to the specific elements within the glp-1 3' untranslated region (3' UTR). When POS-1 or GLD-1 binding is disrupted by mutation of their respective elements, the expression pattern of a glp-1 3' UTR transgene changes in both the germline and in embryos. The mechanism by which POS-1 and GLD-1 mediate translation repression is not well understood. Previous work showed that loss of POS-1 reduces the average polyA tail length of endogenous glp-1 transcripts in embryos. Here, we show that mutation of either the GLD-1 or POS-1 binding motifs in transgenic reporters does not change polyA site selection, and has no effect on polyA tail length distribution, in young adults. These results rule out alternative polyA site usage as a mechanism of regulation, and suggest that the decrease previously observed in endogenous glp-1 polyA tail lengths upon POS-1 RNAi might be indirect. We will expand our analysis by measuring reporter mRNA abundance, polysome association, and polyA tail length distribution in embryos and young adults. When complete, our studies will provide insights of into the mechanism of glp-1 translational regulation in C. elegans germline.

Huggins, H. et al. (2019) International Worm Meeting "C. elegans mRNA cap-binding proteins IFE-1 and IFE-3 have divergent roles in germ cell development."

Subash, J., Hoffman, J., Huggins, H., Stoffel, H., Lee, MH., Keiper, B.

[International Worm Meeting, 2019]

The last step of gene expression, protein synthesis, converts biological information into function. Particularly in germ cells, where mRNA translational control modulates most gene expression, protein synthesis drives cellular function and fate. A current model suggests that the spatial and temporal translation of select mRNAs is determined by a remodeling of complexes that contain RNA-binding proteins, miRNAs, and translation initiation factors. We have uncovered discrete developmental roles for three translation initiation factor eIF4E isoforms (IFE-1, -2, and -3) in C. elegans germ cells. All IFEs bind the m7GTP caps of mRNAs, however each appears to recruit a distinct subset of mRNAs to ribosomes. Individual mutations in ife genes have very different phenotypes, suggesting that over evolutionary time they have gained divergent functions. Here, we address the divergent roles of IFE-1 and IFE-3 in germ cell sex-determination and gamete development. Hermaphrodites generate a defined number of sperm during larval stages, then during adulthood germ cells switch their spermatogenic program to one of oogenesis. This switch is largely accomplished via translational control. We found that IFE-3, is critical for germ cell sex-determination. Hermaphrodites deficient in IFE-3 produced sperm continuously throughout life. Beyond its roles in germ cell sex-determination, IFE-3 is important for the completion of oocyte meiosis and growth. By contrast, IFE-1 is essential for spermatogenesis. Worms deficient in IFE-1 display arrested spermatocytes prior to the final cytokinesis event of meiosis. ife-1 mutants also show mild defects in oocyte maturation. Our model is that IFE-1 and IFE-3 exert translational control over select subsets of mRNAs in the germline which contributes to their null mutant phenotypes. We are using combination of reverse genetics, epistasis, protein biochemistry, CRISPR/cas9 genomic editing, polysome profiling, and bioinformatics to determine how each IFE contributes to germ cell fate development.

Olsen PH et al. (1995) International C. elegans Meeting "The post-transcriptional regulation of lin-14 by lin-4 - polysome analysis."

Ambros VR, Olsen PH

[International C. elegans Meeting, 1995]

lin-14 activity is temporally down-regulated during wild type larval development by the action of a small lin-4 antisense RNA. To investigate the mechanism by which lin-4 is regulating Lin-14 protein levels, the distribution of lin-14 mRNA on size-fractionated polysomes was analyzed. If lin-4 inhibits lin-14 at the level of translational initiation in the late L1 and L2 stages, we would expect to find a decrease in the fraction of large lin-14 RNA-containing polysomes. The principal result is that there is no change in the size distribution of lin-14 polysomes between the L1, when Lin-14 protein is abundant, and the L2, when the steady-state level of Lin-14 protein is essentially undetectable. The second result is that the 22 nucleotide lin-4 RNA is detected in polysome fractions in the approximate molar ratio of 5 to 12 lin-4 RNA's per lin-14 mRNA. To test whether the cosedimentation of lin-4 RNA with lin-14 is specific to the presence of the lin-14 3'UTR, polysomes from C. elegans extracts containing a lin-14 (n355n679) allele that reportedly lacks putative lin-4 binding sites were fractionated. lin-4 RNA was also found to cosediment with this lin-14 (n355n679), though at an apparently lower stoichiometry. Taken together, these results suggest that either Lin-14 protein is made in the L2 but is tagged for rapid degradation, a process signaled by lin-4 RNA or alternatively, the lin-14 polysomes may no longer be translationally competent in the presence of lin-4 RNA. Furthermore, lin-4 RNA may not only be associatedwith lin-14 RNA but also other RNA's or the translational machinery. Experiments are underway to examine these issues.

Keiper, Brett D et al. (2021) International Worm Meeting "Isoforms of eIF4G (ifg-1) are differentially expressed to modulate mRNA translation initiation mechanism in development."

Hoffman, Jenna L, Kim, Eun Suk, Keiper, Brett D

[International Worm Meeting, 2021]

Cellular identity is critically determined by new protein synthesis in cells. Oocytes, spermatocytes and early embryos use predominantly mRNA translational control to regulate in spatial and temporal gene expression rather than transcription. Repression of mRNAs by RNA binding proteins and licensing by miRNAs have been extensively studied in C. elegans germ cells. But there is far less known about the positive translation mechanisms that overcome repression. Positive regulation is mediated by the translational machinery, eIF4 factors, that recruit mRNAs to ribosomes. In recent years we have shown that recruitment, like repression, is an mRNA-selective process. Translation factor eIF4G represents the core of the mRNA recruiting complex. In all organisms there are (at least) two major forms of eIF4G that utilize different mechanisms: one brings together a complex that recognizes the m7GTP mRNA cap (cap-dependent, CD), and the other builds a complex that recruits without cap recognition (cap-independent, CI). Worms express these two isoforms [p170 (CD) and p130 (CI)] from a single ifg-1 gene. Here we show by fluorescent tagging of isoforms in vivo that the CD and CI isoforms vary greatly in abundance during late oogenesis and in early embryos. Using ifg-1 mutants and isoform RNAi we demonstrate that the CD:CI isoform balance plays a critical role in preventing germ cell apoptosis but is largely dispensable following fertilization. The exception is for P granule localization, where suppression of the CD isoform promotes ectopic P granules in early embryos. A Polysome-Seq survey identified a subset of mRNAs that preferentially translates by CI mechanism, a subset that strongly favors CD mechanism, and many that show little preference. Our findings suggest that germ cells and embryos intentionally increase and decrease the CD and CI eIF4G isoforms to regulate mRNA translation by altering initiation mechanisms.

Melissa Richardson et al. (2008) Development & Evolution Meeting "Protein Synthesis in the Germline: The Role of IFE-1"

Elizabeth Cronland, Melissa Richardson, Susan Strome, Brett Keiper

[Development & Evolution Meeting, 2008]

Fertility and embryonic viability are measures of efficient germ cell growth and differentiation. During oogenesis, spermatogenesis and embryogenesis cells initially proliferate then differentiate into specific tissues. New proteins are required for both cell growth and differentiation, requiring qualitative and quantitative changes in protein synthesis. During late gametogenesis and early embryogenesis the expression of the appropriate proteins is a primary function of translational control. Translational control of mRNAs is mediated in part by eukaryotic initiation factor 4E (eIF4E). eIF4E binds the methylated 5' cap of mRNA and recruits it to the ribosome. C.elegans encodes five isoforms of eIF4E (termed IFE-1-5). IFE-1 is expressed primarily in the germline and is the only isoform that associates with P granules by binding directly to PGL-1. P granules also contain stored mRNAs needed for oogenesis and early embryogenesis. Using a strain that lacks IFE-1, we assessed the translational efficiency of maternal mRNAs bound and not bound to P granules by polysome fractionation. Translation of pos-1, pal-1, mex-1, oma-1 and glp-1 mRNA was inefficient in the ife-1(-/-) strain relative to wild type worms. GAPDH mRNA translation was not effected. We also observed differences in the expression of their products, in particular, MEX-1 during oogenesis. In males, secondary spermatocytes fail to complete late stages of maturation in absence of IFE-1. Preliminary evidence shows these immature spermatocytes induce CED-4/Apaf-1 expression, suggesting they undergo apoptosis. In ife-1(-/-) worms fertility was decreased by 80% due to decreased viability of both oocytes and spermatocytes. Our data indicates an essential role for eIF4E (IFE-1 isoform) in late oogenesis and spermatogenesis. We suggest IFE-1 preferentially recruits regulated mRNAs at critical times during germ cell development (Supported by NSF Grant MCB-0321017 to BDK).