专访RNAi前沿科学家刘一 《Nature》封面文章作者

　　

生物通编者按：2009，已渐行渐远，生命科学的精彩还在继续演绎。一个新的发现可能创造一个全新的研究领域，一个新的发现可能彻底颠覆经典理论，每一位科学家都是生命科学领域的弄潮儿，他或推动着生命科学深刻地变革，或默默填补生命科学认识的鸿沟。2009年，点亮生命之光的是谁？奏响生命之歌的是谁？关注他们，关注赛默飞世尔特约之2009年度生命科学十大风云人物评选！

 

5月14日的《Nature》杂志以封面文章形式发表了德克萨斯大学西南医学中心刘一的一篇RNAi研究前沿文章，刘一教授发现了一类新的RNAi分子，qiRNA。人们对RNAi的了解又深入了一步。

 

为了让读者更进一步的了解qiRNA，生物通记者采访了文章的通讯作者刘一教授，百忙中的刘一教授爽快的接受了我们的采访。

 

目前，刘一任德克萨斯大学西南医学中心生理系教授，他的实验室主要从事生物昼夜节律分子机理与RNAi两方面的研究。

 刘一教授近照

 

以下就qiRNA的问题采访刘一教授。

 

生物通：您发现的这类qiRNA与我们常规的siRNA有什么样的区别？

 

刘一：脉孢菌的qiRNAs与siRNA在大小上不同，相比siRNA要小2-3nts，其产生机制和发挥的生物功能也与siRNA不一样。合成qiRNA，首先要以单链RNA（single-stranded RNA,ssRNA）为模板，这类模板被称为变体RNA（aberrant RNAs，aRNAs）。aRNA能被依赖于RNA的RNA聚合酶（RdRPs）特异性识别，在酶的作用下合成双链RNA（double stranded RNA，dsRNA）。然而，aRNA具体的合成过程以及如何被RdRPs识别，这些都是未知的。

 

生物通：qiRNA的发现有什么重要的意义？

 

刘一：我们的研究小组的研究取得了两个重要的成果，我们首次发现了DNA损伤与RNAi路径的特殊关联。qiRNA的合成由DNA损伤诱导启动，DNA损伤会启动损伤应答，合成qiRNA抑制蛋白翻译。我们发现在DNA重组和转座子转座过程中自发性的DNA损伤会激发DNA重复序列产生多种小RNA（small RNA）。

 

其次，我们的研究成果对小RNA和aRNA的生物合成的研究具有重要的意义。在研究qiRNA的过程中，我们发现合成qiRNA首先需要有DNA损伤诱导的aRNA做模板。令人惊讶的是，常规的依赖DNA的RNA聚合酶（polⅠ,Ⅱ&Ⅲ）并不能用来转录aRNA，这表明aRNA的合成过程依赖一种新的依赖DNA的RNA聚合酶（DNA-dependent RNA polymerases，DdRP）。先前的研究认为，DdRP QDE-1是在aRNA合成后才发挥功能，而我们的研究发现QDE-1其实在aRNA的合成前期发挥重要的作用。并且，我们还发现QDE-1不仅是RdRP，还是一种依赖DNA的RNA聚合酶（DNA-dependent RNA polymerases，DdRP）。它首先用其DdRP的活性转录aRNA，接着用其RdRP的活性合成dsRNA。自aRNA被QDE-1合成后，其优先地被QDE-1识别合成dsRNA，我们的研究模型正好完美的解释了这一现象。

 

生物通：以真菌为研究模型，所得出的结论能推及到高等生命吗？

 

刘一：其实，很多人都不知道脉孢菌其实是首批用来研究RNAi的系统之一。实际上，RdRP QDE-1是最早鉴定的与RNAi有关的基因（1999）。此外，RecQ helicase QDE-3和Argonaute protein QDE-2紧接着就被发现了（分别在1999和2000年被发现）。显然的，脉孢菌是研究RNAi的生物模型，它具有遗传学和生化操作学方面的优势，我们用脉孢菌研究得出的结论对研究高等真核生物的RNAi机制具有重要的指导意义。

 

生物通：在您的研究过程中，主要采用的技术有哪些？最关键的技术要点在哪？

 

刘一：我们主要用的还是常规的技术，不过，我们有将生化，分子生物学和遗传学技术有机地结合起来解决一些关键的问题。

 

生物通：对于qiRNA，未来您的研究计划有哪些？

 

刘一：未来，我们将继续研究qiRNA的功能和发生机制。

 

生物通：您能谈谈，RNAi领域未来研究的趋势吗？很多科学家都认为RNAi具有极其广阔的临床应用前景，您有什么看法，要走向临床，RNAi还有哪些困难要克服？

 

刘一：目前RNAi领域在集中研究Agarnaute binding small RNAs和它们的功能，其他小RNA和非编码RNA其实在诸多生物过程中起关键的作用，这是个巨大的课题，还有很多的结构功能以及调节机制没有被披露。

 

我认为，在未来RNAi将完全改变人们用药的方式，一旦找到完善的RNAi传递方式就可以完成这一梦想。在走向临床方面，主要的障碍在于治疗性RNAi的传递系统。只有克服这一难题才可能将RNAi技术应用于临床。

 

生物通：RNAi领域可以说是一个迅速发展起来的学科，要保持走在前沿，您觉得对科学研究工作者有哪些要求？如何做到不断创新，突破？青年学子应该如何培养科学思维？

 

要走在前沿，我觉得需要不断地去发现了解重要的生物学问题，并且深入持久地挖掘生物学问题背后的机制。

 

不断创新突破的关键在于，保持问题驱动性（question- driven）的探索精神。

 

十分感谢刘一教授在百忙中抽空接受生物通的采访。       

 

刘一简介

 

EDUCATION:

 

8/1990-8/1995               Ph.D. Vanderbilt University, Nashville, TN.  Advisor: Dr. Carl H. Johnson. Dissertation: Molecular mechanism of circadian clock in cyanobacteria.

 

9/1985-7/1989              B.S. Wuhan University, Wuhan, Hubei, China.

 

 

PROFESSIONAL EXPERIENCE:

 

9/2008-present      Professor, University of Texas Southwestern Medical Center, Dallas, TX

 

2008-2013             Editorial Board Member, Journal of Biological Chemistry

 

3/2006-3/2010               Elected Neurospora Policy Committee member. Scientific Chair of the 2008 Neurospora Meeting.

 

7/2005-present               Chairman of the Integrative Biology Graduate Program

 

9/2004                          Associate Professor, University of Texas Southwestern Medical Center, Dallas, TX

 

9/1999-8/2004               Assistant Professor and Louise W. Kahn Endowed Scholar in Biomedical Research and Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX

 

9/1995-9/1999              Postdoctoral Research Associate with Dr. Jay Dunlap,                                          Department of Biochemistry, Dartmouth Medical School, Hanover, NH.

 

9/1990-5/1995         Teaching Assistant, Department of Biology, Vanderbilt University.

 

 

FELLOWSHIPS AND AWARDS:

 

3/2004                          The Beadle and Tatum Award, The Neurospora policy committee. Given to one researcher every two years for outstanding contribution using Neurospora as a model system.

 

1999-2003                     Louise W. Kahn Endowed Scholar in Biomedical Research

 

8/1998-9/1999               NIH National Research Service Award for Individual Postdoctoral Fellows. Grant Number: 1F32 GM19230-01.

 

9/1990-8/1995              Vanderbilt University Graduate Fellowship and Tuition Fellowship.

 

7/1995                          Best Conference Abstract Award, American Physiologist Society

 

9/1994                          Vanderbilt University Ph.D. Dissertation Enhancement Award.

 

 

Current Grants:

 

NIH, R01 GM068496 Posttranscriptional Regulation of the Neurospora Circadian Clock, 8/1/03-7/31/11. Direct cost: $210,000/year.

 

NIH, R01 GM062591 Molecular Mechanism of the Neurospora Circadian Clock, 2/1/01-

1/31/10. Direct cost: $200,000/year.

 

NIH, RO1 GM084283 Double-stranded RNA-mediated signaling pathway and gene silencing. Funding expected 4/1/09. $180,000/year

 

Welch Foundation, Biochemical analysis of COP9 signalosome, 6/1/03-5/31/09. Direct cost: $50,000/year.

 

UT Southwestern High Risk/High Impact grant, 2007-2008. $75,000

Welch Foundation, Biochemical analysis of COP9 signalosome, 6/1/03-5/31/09. Direct cost: $50,000/year.

 

 

Teaching:

 

Chairman of the Integrative Biology Graduate Program, 7/2005-present

 

Co-organizer of the Integrative Biology weekly Works in Progress (9/2005-present)

 

Organizing the Integrative Biology Journal Clubs, 9/2005-present

Currently, there are six interest-based journal clubs (Cancer, Metabolism, Diabetes, CNS Diseases, Cardiac, Human Genetics)

 

Coordinator of the Cells & Organelles literature Critique seession of the DCMB Core course, 2003-present. Responsible for recruiting faculty discussion leaders, selecting papers and providing paper guidelines.

 

Lectured in the Response to Stress course (on How environment entrains circadian clock): 2000-present.

 

Lectured in the Cell signaling course (on the Molecular mechanism of circadian clock): 2005-present.

 

 

 

PUBLICATIONS:

 

1. Liu, Y., S.S. Golden, T. Kondo, M. Ishiura, and C.H. Johnson. (1995) Bacterial luciferase as a reporter of circadian gene expression in cyanobacteria.  J. Bacteriology 177:2080-2086.

 

2. Liu, Y., N.F. Tsinoremas, C.H. Johnson, N.V. Lebedeva, S.S. Golden, M. Ishiura, and T. Kondo. (1995) Circadian orchestration of gene expression in cyanobacteria. Genes & Development  9:1469-1478.

 

3. Liu , Y. and N.F. Tsinoremas. (1996) Unusual gene arrangement for the putative chromosome replication origin and circadian gene expression of dnaN gene in Synechococcus sp. strain PCC 7942. Gene 172 (1):105-109.

 

4. Liu, Y., N.F. Tsinoremas, S.S. Golden, T. Kondo, and C.H. Johnson. (1996) Circadian expression of genes involved in the de novo purine biosynthetic pathway of cyanobacteria.  Molecular Microbiology  20 (5): 1071-1081.

 

5. Garceau, N.C., Y. Liu, J.J. Loros, and J.C. Dunlap. (1997) Alternative initiation of translation and time-specific phosphorylation yield multiple forms of the essential clock protein FREQUENCY. Cell 89:469-476

 

6. Liu, Y., N.Y. Garceau, J.J. Loros, and J.C. Dunlap. (1997) Thermally regulated translational control of the frequency protein mediates aspects of temperature responses in the Neurospora circadian clock.  Cell 89: 477-486. (subject of a mini review in Cell).

 

7. Dunlap, J. C., J.J. Loros, S.K.Crosthwaite, Y. Liu  et al.,  (1998) The circadian regulatory system in Neurospora. In Light and Time in Microbial Systems, D. Roberts, ed. (Cambridge: Cambridge Univ. Press).

 

8. Liu, Y., M. Merrow, J.J. Loros, and J.C. Dunlap. (1998) How temperature changes reset a circadian oscillator.  Science 281:825-829.

 

9. Dunlap, J.C., J.J. Loros, Y. Liu, and S.K. Crosthwaite(1999) Eukaryotic circadian systems: cycles in common.  Genes to Cells 4:1-10.

 

10. Liu, Y., C. Heintzen, J.J. Loros, and J.C. Dunlap. (1999) Regulation of clock genes. Cell. Mol. Life Sci. 55:1195-1205.

 

11. Liu, Y., J.J. Loros, and J.C.Dunlap (2000). Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock. Proc. Natl. Acad. Sci. USA 97: 234-239.

 

12. Cheng, P, Y. Yang, C. Heintzen, and Y. Liu (2001) Coiled-coil domain mediated FRQ-FRQ interaction is essential for its circadian clock function in Neurospora.  EMBO J.  20: 101-108.

 

13. Cheng, P, Y. Yang, and Y. Liu. (2001) Interlocked feedback loops contribute to the robustness of the Neurospora circadian clock. Proc. Natl. Acad. Sci. USA 98:7408-7413

 

14. Yang, Y., P. Cheng, G. Zhi, and Y. Liu (2001) Identification of a calcium/calmodulin-dependent protein kinase that phosphorylates the Neurospora circadian clock protein FREQUENCY. J. Biol. Chem. 276: 41064-41072

 

15. Cheng, P, Y. Yang, K. H. Gardner, and Y. Liu. (2002)  PAS domain mediated WC-1/WC-2 heterodimeric complex is essential for their circadian clock function and the stability of WC-1. Mol. Cell. Biol  22: 517-524

 

16. Yang, Y., P. Cheng, and Y. Liu. (2002) Regulation of the Neurospora circadian clock by casein kinase II. Genes & Development  15:994-1006.

 

17. He, Q.*, P. Cheng*, Y. Yang, L. Wang, K. H. Gardner, and Y. Liu. (2002)  White Collar-1, a DNA-binding transcription factor and a light sensor. Science. 297: 840-843 (cover article, accompanied by a Perspective)

 

18. Froehlich, A. C., Y. Liu, J. J. Loros, and J. C. Dunlap.  (2002) White Collar-1, a circadian blue light photoreceptor, binds to the frequency promoter. Science. 297: 815-819 843 (cover article, accompanied by Perspective)

 

19. Cheng, P, Y. Yang, L, Wang, Q. He, and Y. Liu. (2003) WHITE COLLAR-1, a multifunctional Neurospora protein involved in the circadian feedback loops, light sensing, and transcription repression of wc-2.  J. Biol. Chem.  278:3801-3808.

 

20. Cheng, P., Q. He, Y. Yang, L, Wang, and Y. Liu. (2003)  Functional conservation of light, oxygen, or voltage domains in light sensing.  Proc. Natl. Acad. Sci. USA 100:5938-5943

 

21. Liu, Y., (2003) J. Biol. Rhythms  Molecular mechanisms of entrainment of the Neurospora circadian clock.  18: 195-205.

 

22. Yang, Y., P. Cheng, Q. He, L. Wang, and Y. Liu  (2003) Phosphorylation of FREQUENCY Protein by Casein Kinase II is Necessary for the Function of the Neurospora Circadian Clock.  Mol. Cell. Biol  23:6221-6228

 

23. He, Q.*, Cheng, P.*, Y. Yang, Q. He, H. Yu, and Y. Liu. (2003) FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation.  EMBO J. 22:4421-4430

 

24. Liu, Y., Q. He, and P. Cheng. (2003) Photoreception in Neurospora, A Tale of Two White Collar Proteins.   Cell. Mol. Life Sci.  60:2131-2138.

 

25. Yang, Y., Q. He, P. Cheng, P. Wrage, Oded Yarden, and Y. Liu. (2004) Distinct roles for PP1 and PP2A in the Neurospora circadian clock. Genes & Development. 18:255-260

 

26. Min, H., Y. Liu, C. H. Johnson, and S. S. Golden. (2004) Phase Determination of Circadian Gene Expression in Synechococcus elongatus PCC 7942.  J. Biol. Rhythms  19:103-112.

 

27. Liu, Y., Y. Yang. and Q. He (2005) Posttranscriptional regulation of the Neurospora circadian clock. In “Circadian Clocks in Eukaryotic Microbes”. Edited by Fred Kippert. published by Landes Bioscience. (http://www.eurekah.com/abstract.php?chapid=2329&bookid=180&catid=85&PHPSESSID=4ca3afca5fc5f5d6da4e00fcc7abc154)

 

28. Liu, Y.(2005)  Analysis of posttranslational regulations in the Neurospora circadian clock. Methods in Enzymology. “Circadian Rhythms”,Edited by Michael Young.  393:379-393

 

29. Cheng, P., Q. He, Q. He, L. Wang, and Y. Liu.  (2005) Regulation of the Neurospora circadian clock by an RNA helicase. Genes & Development  19: 234-241.

 

30. He Q., H. Shu, P. Cheng, S. Chen, L. Wang, and Y. Liu. (2005) Light-independent phosphorylation of WHITE COLLAR-1 is important for its role in the circadian negative feedback loop.  J. Biol. Chem. 280: 17526-17532.

 

31. He, Q., Cheng, P., Q. He, and Y. Liu. (2005) The COP9 signalosome regulates the Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex.  Genes & Development 19: 1518-1531.

 

32. He, Q, and Y. Liu (2005) Degradation of the Neurospora circadian clock protein FREQUENCY through the ubiquitin-proteasome pathway. Biochem Soc Trans. 33:953-956.

 

33. He, Q. and Y. Liu (2005) Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation. Genes & Development 19: 2888-2899.

 

34. Huang, G., L. Wang, and Y. Liu (2006) Molecular mechanism of suppression of circadian rhythm by a critical stimulus. EMBO J.  25: 5349-5357.

 

35. Liu. Y. and D. Bell-Pedersen (2006) Circadian rhythms in Neurospora and other filamentous fungi. Eukaryotic Cell. 5: 1184-1193.

 

36. He, Q., J. Cha, Q. He, H. Lee, Y. Yang, and Y. Liu. (2006) CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop. Genes & Dev. 20:2552-2565.

 

37. Heintzen, C. and Y. Liu (2007) The Neurospora crassa circadian clock. Adv in Genetics, 58:25-66

 

38. Maiti, M., HC. Lee, and Y. Liu (2007) QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands. Genes & Dev. 21: 590-600

 

39. Choudhary, S.*, HC Lee*, M. Maiti*, Q. He, P. Cheng, Q. Liu, and Y. Liu (2007) A double-stranded RNA response program important for RNAi efficiency. Mol. Cell. Biol. 27:3995-4005. (the cover article).

 

40. Guocun Huang, She Chen, Shaojie Li, Joonseok Cha, Chengzu Long, Lily Li, Qiyang He and Yi Liu. (2007) Protein Kinase A and Casein Kinases Mediate Sequential Phosphorylation Events in the Circadian Negative Feedback Loop. Genes & Dev. 21: 3283-3295.

 

41. Cha, J., G. Huang, J. Guo, and Y. Liu (2007) Posttranslational control of the Neurospora circadian clock. Cold Spring Harbor Symposia on Quantitative Biology 72: 185-91.

 

42. Cha J, Chang SS, Huang G, Cheng P, and Y. Liu (2008) Control of WHITE COLLAR localization by phosphorylation is a critical step in the circadian negative feedback process. EMBO J. 27: 3246-3255.

 

43. He Q., Tang C, and Y. Liu (2008) The roles of the ubiquitin-proteasome system in the control of circadian clocks. The Ubiquitin Proteasome System. Edited by Napoli and Wojcik. In press.

 

44. Jinhu Guo, Guocun Huang, Joonseok Cha, Yi Liu (2008) Protein analyses of the filamentous fungus Neurospora crassa. Molecular and Cell Biology Methods for Fungi. Edited by Amir Sharon. In press.

 

45. Lee HC, Chang SS, Choudhary S, Aalto AP, Maiti M, Bamford DH, Y. Liu. (2009) qiRNA is a new type of small interfering RNA induced by DNA damage. Nature 459(7244):274-277.

 

46. Chi-Tai Tang, Shaojie Li, Chengzu Long,  Joonseok Cha, Guocun Huang, Lily Li, She Chen and Yi Liu (2009) Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events. Proc. Natl. Acad. Sci. USA (in press).