Loss of flavin adenine dinucleotide (FAD) impairs sperm function and male reproductive advantage in C. elegans

Yen, Chia‐An; Ruter, Dana L.; Turner, Christian D.; Pang, Shijin; Curran, Sean P.

doi:10.7554/elife.52899

Cited by 31 publications

(29 citation statements)

References 72 publications

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“…10-15 males were dissected in 30uL of 500μg/mL Pronase E (Millipore Sigma), which was dissolved in sperm medium (50 mM HEPES, 50 mM NaCl, 25 mM KCl, 5 mM CaCl2, 1 mM MgSO4, 1 mg/ml BSA), as described previously (S. Ward et al, 1983; Yen et al, 2020). Spermatids were incubated for 15 minutes at room temperature in a humid chamber before mounting and imaging on a DeltaVision Elite microscope (GE Healthcare) using a 60x N.A.…”

Section: Methods Detailsmentioning

confidence: 99%

Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1

Nguyen

Phillips

2020

Preprint

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1CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs 2 targeting most germline-expressed genes. Here we show that the two isoforms of CSR-3 1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the 4 germline and during all stages of development while CSR-1A expression is restricted to 5 germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates 6 preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas 7 CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. 8 Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine 9 modifications, which are necessary for the preferential binding of CSR-1A to 10 spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory 11 mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA 12 binding between similar Argonaute proteins with overlapping temporal and spatial 13 localization.14 15 84 binding to both spermatogenic and oogenic siRNAs. Thus, in this study, we have 85 identified the first instance of methylarginine modification of a C. elegans Argonaute 86 protein and demonstrated a previously unappreciated mechanism by which Argonaute 87 proteins can acquire small RNA specificity. 88 89 Results 90The long isoform of CSR-1 is selectively expressed during spermatogenesis 91 The Argonaute protein, CSR-1, has two isoforms, though previously little was known 92 about their differential expression or function. The longer isoform, referred to as CSR-93 5 1A, and the shorter isoform, CSR-1B, share complete sequence homology, except for a 94 unique exon at the 5' end of CSR-1A ( Figure 1A). Both isoforms are expressed, though 95 at differential levels ( Figure 1B) (Claycomb et al., 2009; Gerson-Gurwitz et al., 2016). To 96 explore the distinct functions of the two CSR-1 isoforms, we used CRISPR/Cas9 to 97 endogenously tagged CSR-1A at its N-terminus with a 2xHA/mCherry tag. We also 98 tagged both CSR-1A and CSR-1B in the same strain by adding a 3xFLAG/GFP tag to 99 the N-terminus of CSR-1B ( Figure 1A). When both isoforms are tagged, CSR-1A+B is 100 strongly expressed throughout the germline and during all developmental stages, in 101 agreement with previous studies ( Figure 1A) (Claycomb et al., 2009). Interestingly, 102 when only CSR-1A is tagged, expression is restricted to the spermatogenesis region of 103 the germline during the fourth larval stage (L4) of hermaphrodites and in males ( Figure 104 1A and S1). We confirmed this observation by western blot analysis of all larval stage 105 and adult animals, detecting CSR-1A expression robustly during the L4 stage and very 106 weakly in gravid adults, coinciding temporally with spermatogenesis in C. elegans 107 ( Figure 1C). We next examined the differences in csr-1a and csr-1b transcript levels by 108 performing RT-qPCR on wild-type animals using primers that recognize either csr-1a 109 uniquely, or csr-1a and csr-1b together. We observed that the csr-1a transcript ...

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Section: Methods Detailsmentioning

confidence: 99%

Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1

Nguyen

Phillips

2020

Preprint

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“…S3a). Interestingly, while some physiological responses, including reproduction, are sensitive to diet type 8,16,35,[38][39][40][41][42][43] , the reduced size phenotype in mafr-1 (KO) males is also observed on the HT115 E.coli K-12 bacterial diet ( Fig. S3b-c).…”

Section: Mafr-1 Regulates Male Sperm Fitnessmentioning

confidence: 97%

“…In order to test the physiological consequence of mafr-1 (KO) on sperm fitness phenotypes, we designed an assay to assess sperm quality 35 . When hermaphrodites are mated to WT males, nearly all resulting progeny are derived from male sperm 26 .…”

Section: Mafr-1 Regulates Male Sperm Fitnessmentioning

confidence: 99%

“…In addition to size, the speed of male sperm is greater than hermaphrodite-derived sperm, which also enhances male competitive advantage for fertilizing an oocyte 24 . To become motile, inactive spermatids must become activated by developing pseudopodia, a sophisticated process under the control of genetic and environmental factors 32,34,35 . In the laboratory, isolated spermatids can be activated in vitro by exposure to Pronase 34,35 .…”

mentioning

confidence: 99%

“…To become motile, inactive spermatids must become activated by developing pseudopodia, a sophisticated process under the control of genetic and environmental factors 32,34,35 . In the laboratory, isolated spermatids can be activated in vitro by exposure to Pronase 34,35 . RNAi of these potential interactors resulted in no significant change in activation (Fig.…”

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confidence: 99%

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Roles for the RNA polymerase III regulator MAFR-1 in regulating sperm quality in Caenorhabditis elegans

Hammerquist¹,

Curran²

2020

Sci Rep

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The negative regulator of RNA polymerase (pol) III mafr-1 has been shown to affect RNA pol III transcript abundance, lipid biosynthesis and storage, progeny output, and lifespan. We deleted mafr-1 from the Caenorhabditis elegans genome and found that animals lacking mafr-1 replicated many phenotypes from previous RNAi-based studies and discovered a new sperm-specific role. Utilizing a yeast two-hybrid assay, we discovered several novel interactors of MAFR-1 that are expressed in a sperm- and germline-enriched manner. In support of a role for MAFR-1 in the male germline, we found mafr-1 null males have smaller spermatids that are less capable in competition for fertilization; a phenotype that was dependent on RNA pol III activity. Restoration of MAFR-1 expression specifically in the germline rescued the spermatid-related phenotypes, suggesting a cell autonomous role for MAFR-1 in nematode male fertility. Based on the high degree of conservation of Maf1 activity across species, our study may inform similar roles for Maf1 and RNA pol III in mammalian male fertility.

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Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans‐4‐hydroxy‐L‐proline

et al. 2021

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Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD + -dependent oxidations of L-glutamate-γ-semialdehyde to L-glutamate (proline metabolism), and 4-hydroxy-L-glutamate-γ-semialdehyde to 4-erythro-hydroxy-L-glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4-hydroxyproline using steady-state kinetics and X-ray crystallography. Trans-4-hydroxy-L-proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L-proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L-stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans-4-hydroxy-L-proline and trans-4-hydroxy-D-proline. The structures suggest that the 10-fold greater preference for the L-stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans-4-hydroxy-L-proline. In contrast, the amine group of the D-stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans-4-hydroxy-L-proline, analogous to the known inhibition of proline catabolism by L-proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans-4-hydroxy-L-proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.

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Loss of flavin adenine dinucleotide (FAD) impairs sperm function and male reproductive advantage in C. elegans

Cited by 31 publications

References 72 publications

Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1

Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1

Roles for the RNA polymerase III regulator MAFR-1 in regulating sperm quality in Caenorhabditis elegans

Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans‐4‐hydroxy‐L‐proline

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