Adhvaryu Keyur, Firoozi Ghazaleh, Motavaze Kamyar, Lakin-Thomas Patricia
Dalla Lana School of Public Health, University of Toronto, Toronto, Ontario M5T 1R4, Canada.
J Biol Rhythms. 2016 Jun;31(3):258-71. doi: 10.1177/0748730416639717. Epub 2016 Mar 29.
The circadian rhythms found in almost all organisms are driven by molecular oscillators, including transcription/translation feedback loops (TTFLs). However, TTFL-independent oscillators can drive rhythms in both eukaryotes and prokaryotes. The fungus Neurospora crassa is a model organism for studying the molecular mechanism of the circadian clock. Although a circadian TTFL involving the proteins FRQ, WC-1, and WC-2 is well-characterized in N. crassa, rhythms can still be observed in the absence of this feedback loop. These rhythms are said to be driven by 1 or more FRQ-less oscillator(s) (FLOs). The prd-1 mutation lengthens the period in frq wild type and was previously shown to severely disrupt FRQ-less rhythms in frq null mutants under several different conditions; therefore, the prd-1 gene product is a candidate for a component of a FLO. We report here that prd-1 also disrupts free-running rhythms in wc-1 null mutants, confirming its effects on FRQ-less rhythms. We have now mapped and identified the prd-1 gene as NCU07839, a DEAD-box RNA helicase dbp-2 Complementation with the wild-type gene corrects the rhythm defects of the prd-1 mutant in the complete circadian system (when the FRQ-based TTFL is intact) and also the free-running FRQ-less rhythm on low choline. A PRD-1-GFP fusion protein localizes to the nucleus. The prd-1 mutant has a single base pair change in the first base of an intron that results in abnormally spliced transcripts. FRQ-less rhythms on low choline, or entrained to heat pulses, were only marginally affected in strains carrying deletions of 2 other RNA helicases (prd-6 and msp-8). We conclude that PRD-1 is a member of an RNA helicase family that may be specifically involved in regulating rhythmicity in N. crassa in both the complete circadian system and FLO(s).
几乎在所有生物体中发现的昼夜节律是由分子振荡器驱动的，包括转录/翻译反馈环（TTFLs）。然而，不依赖TTFL的振荡器也能在真核生物和原核生物中驱动节律。真菌粗糙脉孢菌是研究生物钟分子机制的模式生物。虽然在粗糙脉孢菌中，一个涉及FRQ、WC-1和WC-2蛋白的昼夜TTFL已得到充分表征，但在没有这种反馈环的情况下仍能观察到节律。这些节律据说是由1个或多个无FRQ振荡器（FLOs）驱动的。prd-1突变延长了frq野生型的周期，并且先前已表明在几种不同条件下，该突变会严重破坏frq缺失突变体中的无FRQ节律；因此，prd-1基因产物是FLO的一个组成成分的候选者。我们在此报告，prd-1也会破坏wc-1缺失突变体中的自主节律，证实了其对无FRQ节律的影响。我们现已将prd-1基因定位并鉴定为NCU07839，即一种DEAD盒RNA解旋酶dbp-2。用野生型基因进行互补可纠正prd-1突变体在完整昼夜节律系统（当基于FRQ的TTFL完整时）中的节律缺陷，以及在低胆碱条件下的自主无FRQ节律。PRD-1-GFP融合蛋白定位于细胞核。prd-1突变体在内含子的第一个碱基处有一个单碱基对变化，导致转录本异常剪接。在携带另外两种RNA解旋酶（prd-6和msp-8）缺失的菌株中，低胆碱条件下或受热脉冲诱导的无FRQ节律仅受到轻微影响。我们得出结论，PRD-1是RNA解旋酶家族的一员，可能在完整昼夜节律系统和FLOs中特异性参与调节粗糙脉孢菌的节律性。
