Defining the contribution of microRNA-specific Argonautes with slicer capability in animals.

microRNAs regulate gene expression through interaction with an Argonaute protein. While some members of this protein family retain an enzymatic activity capable of cleaving RNA molecules complementary to Argonaute-bound small RNAs, the role of the slicer residues in the canonical microRNA pathway is still unclear in animals. To address this, we created Caenorhabditis elegans strains with mutated slicer residues in the endogenous ALG-1 and ALG-2, the only two slicing Argonautes essential for the miRNA pathway in this animal model. We observe that the mutation in ALG-1 and ALG-2 catalytic residues affects overall animal fitness and causes phenotypes reminiscent of miRNA defects only when grown and maintained at restrictive temperature. Furthermore, the analysis of global miRNA expression shows that the slicer residues of ALG-1 and ALG-2 contribute differentially to regulate the level of specific subsets of miRNAs in young adults. We also demonstrate that altering the catalytic tetrad of those miRNA-specific Argonautes does not result in any defect in the production of canonical miRNAs. Together, these data support that the slicer residues of miRNA-specific Argonautes contribute to maintaining levels of a set of miRNAs for optimal viability and fitness in animals particularly exposed to specific growing conditions.

Defining the contribution of microRNA-specific slicing Argonautes in animals.

microRNAs regulate gene expression through interaction with an Argonaute protein family member. While some members of this protein family retain an enzymatic activity capable of cleaving RNA molecules complementary to Argonaute-bound small RNAs, the role of the slicing activity in the canonical microRNA pathway is still unclear in animals. To address the importance of slicing Argonautes in animals, we created Caenorhabditis elegans strains, carrying catalytically dead endogenous ALG-1 and ALG-2, the only two slicing Argonautes essential for the miRNA pathway in this animal model. We observe that the loss of ALG-1 and ALG-2 slicing activity affects overall animal fitness and causes phenotypes, reminiscent of miRNA defects, only when grown and maintained at restrictive temperature. Furthermore, the analysis of global miRNA expression shows that the catalytic activity of ALG-1 and ALG-2 differentially regulate the level of specific subsets of miRNAs in young adults. We also demonstrate that altering the slicing activity of those miRNA-specific Argonautes does not result in any defect in the production of canonical miRNAs. Together, these data support that the slicing activity of miRNA-specific Argonautes function to maintain the levels of a set of miRNAs for optimal viability and fitness in animals particularly exposed to specific growing conditions.

Identification and sequencing of temperature sensitive alleles of the Anaphase Promoting Complex component mat-3 in C. elegans.

The Anaphase Promoting Complex (APC) regulates the transition from metaphase to anaphase during cell division and is important to prevent defects in chromosome segregation. In a recent temperature sensitive genetic screen looking for further genes involved in fertilization, we isolated a new temperature sensitive allele of mat-3 (as49) . We also sequenced a previously identified mat-3 ( or344 ) allele that did not previously have an annotated sequence. We determined that the as49 allele was an Alanine to Threonine (A451T) mutation in the sixth exon and the or344 mutation was a Leucine to Phenylalanine (L474F) mutation in the seventh exon. These locations of the mutant alleles are consistent with other previously annotated alleles that displayed the same metaphase to anaphase transition defect phenotype and further reinforce the importance of the tetratricopeptide repeats to mediate protein interactions.

SPE-51, a sperm-secreted protein with an immunoglobulin-like domain, is required for fertilization in C. elegans.

Fertilization is a fundamental process in sexual reproduction during which gametes fuse to combine their genetic material and start the next generation in their life cycle. Fertilization involves species-specific recognition, adhesion, and fusion between the gametes.1,2 In mammals and other model species, some proteins are known to be required for gamete interactions and have been validated with loss-of-function fertility phenotypes.3,4 Yet, the molecular basis of sperm-egg interaction is not well understood. In a forward genetic screen for fertility mutants in Caenorhabditis elegans, we identified spe-51. Mutant worms make sperm that are unable to fertilize the oocyte but otherwise normal by all available measurements. The spe-51 gene encodes a secreted protein that includes an immunoglobulin (Ig)-like domain and a hydrophobic sequence of amino acids. The SPE-51 protein acts cell autonomously and localizes to the surface of the spermatozoa. We further show that the gene product of the mammalian sperm function gene Sof1 is likewise secreted. This is the first example of a secreted protein required for the interactions between the sperm and egg with genetic validation for a specific function in fertilization in C. elegans (also see spe-365). This is also the first experimental evidence that mammalian SOF1 is secreted. Our analyses of these genes begin to build a paradigm for sperm-secreted or reproductive-tract-secreted proteins that coat the sperm surface and influence their survival, motility, and/or the ability to fertilize the egg.

Where are all the egg genes?

Complementary forward and reverse genetic approaches in several model systems have resulted in a recent burst of fertilization gene discovery. The number of genetically validated gamete surface molecules have more than doubled in the last few years. All the genetically validated sperm fertilization genes encode transmembrane or secreted molecules. Curiously, the discovery of genes that encode oocyte molecules have fallen behind that of sperm genes. This review discusses potential experimental biases and inherent biological reasons that could slow egg fertilization gene discovery. Finally, we shed light on current strategies to identify genes that may result in further identification of egg fertilization genes.

Sperm fate is promoted by the mir-44 microRNA family in the Caenorhabditis elegans hermaphrodite germline.

Posttranscriptional regulation of gene expression, typically effected by RNA-binding proteins, microRNAs (miRNAs), and translation initiation factors, is essential for normal germ cell function. Numerous miRNAs have been detected in the germline; however, the functions of specific miRNAs remain largely unknown. Functions of miRNAs have been difficult to determine as miRNAs often modestly repress target mRNAs and are suggested to sculpt or fine tune gene expression to allow for the robust expression of cell fates. In Caenorhabditis elegans hermaphrodites, cell fate decisions are made for germline sex determination during larval development when sperm are generated in a short window before the switch to oocyte production. Here, analysis of newly generated mir-44 family mutants has identified a family of miRNAs that modulate the germline sex determination pathway in C. elegans. Mutants with the loss of mir-44 and mir-45 produce fewer sperm, showing both a delay in the specification and formation of sperm as well as an early termination of sperm specification accompanied by a premature switch to oocyte production. mir-44 and mir-45 are necessary for the normal period of fog-1 expression in larval development. Through genetic analysis, we find that mir-44 and mir-45 may act upstream of fbf-1 and fem-3 to promote sperm specification. Our research indicates that the mir-44 family promotes sperm cell fate specification during larval development and identifies an additional posttranscriptional regulator of the germline sex determination pathway.