Animals alter their reproductive programs to accommodate changes in nutrient availability, yet the connections between known nutrient-sensing systems and reproductive programs are underexplored, and whether there is a mechanism that senses nucleotide levels to coordinate germline proliferation is unknown. We established a model system in which nucleotide metabolism is perturbed in both the nematode Caenorhabditis elegans (cytidine deaminases) and its food (Escherichia coli); when fed food with a low uridine/thymidine (U/T) level, germline proliferation is arrested. We provide evidence that this impact of U/T level on the germline is critically mediated by GLP-1/Notch and MPK-1/MAPK, known to regulate germline mitotic proliferation. This germline defect is suppressed by hyperactivation of glp-1 or disruption of genes downstream from glp-1 to promote meiosis but not by activation of the IIS or TORC1 pathways. Moreover, GLP-1 expression is post-transcriptionally modulated by U/T levels. Our results reveal a previously unknown nucleotide-sensing mechanism for controlling reproductivity.

Branched-chain ␣-ketoacid dehydrogenase (BCKDH) catalyzes the critical step in the branched-chain amino acid (BCAA) catabolic pathway and has been the focus of extensive studies. Mutations in the complex disrupt many fundamental metabolic pathways and cause multiple human diseases including maple syrup urine disease (MSUD), autism, and other related neurological disorders. BCKDH may also be required for the synthesis of monomethyl branched-chain fatty acids (mmBCFAs) from BCAAs. The pathology of MSUD has been attributed mainly to BCAA accumulation, but the role of mmBCFA has not been evaluated. Here we show that disrupting BCKDH in Caenorhabditis elegans causes mmBCFA deficiency, in addition to BCAA accumulation. Worms with deficiency in BCKDH function manifest larval arrest and embryonic lethal phenotypes, and mmBCFA supplementation suppressed both without correcting BCAA levels. The majority of developmental defects caused by BCKDH deficiency may thus be attributed to lacking mmBCFAs in worms. Tissue-specific analysis shows that restoration of BCKDH function in multiple tissues can rescue the defects, but is especially effective in neurons. Taken together, we conclude that mmBCFA deficiency is largely responsible for the developmental defects in the worm and conceivably might also be a critical contributor to the pathology of human MSUD.Branched-chain amino acids (BCAAs), 2 leucine, isoleucine, and valine, are essential amino acids that are not only building blocks for protein synthesis but also play important physiological roles (1). Their catabolism is controlled by branched-chain ␣-ketoacid dehydrogenase (BCKDH), a mitochondrial multisubunit enzyme complex (see Fig. 1A). BCKDH is composed of three subunits, E1, E2 and E3, of which E1 and E2 are unique to this complex. The E1 subunit contains two components, E1␣ and E1␤, which in humans are encoded by BCKDHA and BCKDHB, respectively (2, 3). The E2 subunit is encoded by DBT (4). Autosomal recessive mutation in any of these genes results in BCKDH deficiency and causes maple syrup urine disease (MSUD, Online Mendelian Inheritance in Man (OMIM) 248600). Classic MSUD patients have less than 2% BCKDH activity, which results in elevated BCAAs and branched-chain ␣-ketoacids in tissues and plasma. If untreated, patients can develop life-threatening cerebral edema within 10 days of life. MSUD also has a chronic effect on the central nervous system, resulting in dysmyelination and mental retardation in young patients (5, 6).The neurotoxicity of MSUD has been attributed mainly to increased plasma leucine and its derivative, ␣-ketoisocaproic acid (5). They compete with other large neutral amino acids for transportation across the blood-brain barrier by the LAT1 amino acid transporter, causing decreased levels of large neutral amino acids that are precursors for key neuronal factors such as dopamine and serotonin, in the brain in humans (5), classic MSUD mice (7), and rats (8). Increased ␣-ketoisocaproic acid depletes glutamate, affecting transamination in the brain and c...

Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic “enhancer” approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It also suggests that compromising other aspects of gene expression, along with miRISC, can be an effective approach to reveal miRNA functions in specific tissues under specific physiological conditions.

MicroRNAs (miRNAs) are conserved small non-coding RNAs that typically regulate gene expression by binding to the 3' untranslated region (UTR) of mRNAs. Developmental functions of miRNAs have been extensively studied, but additional roles in various cellular processes remain to be understood. The investigation of the biological importance of individual miRNA-target interactions and the miRNA-target interaction network as a whole has been an exciting and challenging field of study. Here we briefly discuss the contributions our lab has made to our understanding of the physiological impact of this miRNA-network in C. elegans, in the context of recent studies in this advancing field. These studies have advanced our knowledge of the role of miRNAs in ensuring a robust cellular response to different physiological conditions. We briefly outline the genetic, biochemical, and computational strategies utilized to understand miRNA functions and discuss our recent study of the miRNA-interaction network in neurons and potential directions for future studies.

The etiology of anemia in adults is often multifactorial. This case highlights an uncommon combination of causes of anemia and the importance of a diagnostic workup guided by a patient's unique history, risk factors, and laboratory findings.