Construction and Functional Study of RagA Transgenic Drosophila

doi:10.13560/j.cnki.biotech.bull.1985.2022-1341

Abstract

Abstract:

Rag GTPase is GTP-binding proteins belonging to a member of the Ras family and localizes on lysosomes. Drosophila RagA protein, a homologue of mammalian RagA/RagB protein, forms a complex with RagC to regulate TORC1 activity. Here we found that knockdown of RagA caused Drosophila development to stagnate at the pupal stage. To explore the function of RagA on Drosophila development, we constructed the overexpressing vector pUASp-RagA-wt with RagA RNA interfering target sites changed. On this basis, the GTP-binding RagA-overexpressed plasmid pUASp-RagA-Q61L and the GDP-binding RagA-overexpressed plasmid pUASp-RagA-T16N were constructed by point mutation. The pUASp-RagA-wt, pUASp-RagA-Q61L and pUASp-RagA-T16N plasmids were screened by microinject and screentransgene lines. The effects of different forms of RagA on the TORC1 activity were evaluated by the method of somatic cell cloning, RagA regulated the TORC1 activity, and it was in the activated state after binding with GTP, and inactivated after binding with GDP. To determine the effects of RagA activity on the Drosophila development under GTP/GDP state, the UASp transgene lines were crossed with Tub-Gal4/TM6 and the eclosion rate of offspring was counted. The results showed that Drosophila with RagA knockdown failed to develop into adults due to obstacles in the pupal stage. The overexpression of wild-type RagA completely avoided the death of Drosophila caused by RagA knockdown, confirming that depletion of RagA was the cause of developmental defects in RagA RNAi. The overexpression of GTP-bound RagA-Q61L partially reduced the lethal effect of Drosophila with RagA knockdown, while the overexpression of GDP-bound RagA-T16N could not decrease the lethal effect of Drosophila with RagA knockdown at all. Our work suggests that RagA plays an important role in the growth and development, and the binding of RagA and GTP/GDP needs to be in a dynamic equilibrium. Binding of RagA only to GTP or GDP would make TORC1 activity too high or too low, thus affecting fruit fly growth and development.

Key words: RagA, microinjection, Drosophila

MENG Guo-qiang, GUAN Jian-wen, NIU Chun-mei, ZHOU Ying, SHEN Su-lin, WEI You-heng. Construction and Functional Study of RagA Transgenic Drosophila[J]. Biotechnology Bulletin, 2023, 39(6): 171-180.

Figures/Tables 10

References 26

[1]	Frasch M. Genome-wide approaches to Drosophila heart development[J]. J Cardiovasc Dev Dis, 2016, 3(2): 20.
[2]	Adams MD, Celniker SE, Holt RA, et al. The genome sequence of Drosophila melanogaster[J]. Science, 2000, 287(5461): 2185-2195. doi: 10.1126/science.287.5461.2185 pmid: 10731132
[3]	Rubin GM, Yandell MD, Wortman JR, et al. Comparative genomics of the eukaryotes[J]. Science, 2000, 287(5461): 2204-2215. pmid: 10731134
[4]	万永奇, 谢维. 生命科学与人类疾病研究的重要模型—果蝇[J]. 生命科学, 2006, 18(5): 425-429.
	Wan YQ, Xie W. Drosophila: an important model organism for understanding basic biological and human disease mechanisms[J]. Chin Bull Life Sci, 2006, 18(5): 425-429.
[5]	Chiang HC, Hodson AC. An analytical study of population growth in Drosophila melanogaster[J]. Ecol Monogr, 1950, 20(3): 173-206. doi: 10.2307/1948580 URL
[6]	Ashburner M, Thompson JN. The laboratory culture of drosophila[M]// AshburnerM, WrightT R F. The genetics and biology of drosophila. 2A. Cambridge: Academic Press, 1980: 1-81.
[7]	Ashburner M, Golic KG, Hawley RS. Drosophila: a laboratory handbook, 2nd ed.[M]// SciTech Book News. New York: Cold Spring Harbor Laboratory Press, 2005: 162-164.
[8]	Bun-Ya M, Harashima S, Oshima Y. Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae[J]. Mol Cell Biol, 1992, 12(7): 2958-2966. doi: 10.1128/mcb.12.7.2958-2966.1992 pmid: 1620108
[9]	Panchaud N, Péli-Gulli MP, de Virgilio C. Amino acid deprivation inhibits TORC1 through a GTPase-activating protein complex for the Rag family GTPase Gtr1[J]. Sci Signal, 2013, 6(277): ra42.
[10]	Binda M, Péli-Gulli MP, Bonfils G, et al. The Vam6 GEF controls TORC1 by activating the EGO complex[J]. Mol Cell, 2009, 35(5): 563-573. doi: 10.1016/j.molcel.2009.06.033 pmid: 19748353
[11]	Kim J, Guan KL. mTOR as a central hub of nutrient signalling and cell growth[J]. Nat Cell Biol, 2019, 21(1): 63-71. doi: 10.1038/s41556-018-0205-1 pmid: 30602761
[12]	Sabatini DM. Twenty-five years of mTOR: Uncovering the link from nutrients to growth[J]. PNAS, 2017, 114(45): 11818-11825. doi: 10.1073/pnas.1716173114 pmid: 29078414
[13]	Rabanal-Ruiz Y, Otten EG, Korolchuk VI. mTORC1 as the main gateway to autophagy[J]. Essays Biochem, 2017, 61(6): 565-584. doi: 10.1042/EBC20170027 pmid: 29233869
[14]	Yu L, Chen Y, Tooze SA. Autophagy pathway: cellular and molecular mechanisms[J]. Autophagy, 2018, 14(2): 207-215. doi: 10.1080/15548627.2017.1378838 pmid: 28933638
[15]	Sancak Y, Peterson TR, Shaul YD, et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1[J]. Science, 2008, 320(5882): 1496-1501. doi: 10.1126/science.1157535 pmid: 18497260
[16]	Sancak Y, Bar-Peled L, Zoncu R, et al. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids[J]. Cell, 2010, 141(2): 290-303. doi: 10.1016/j.cell.2010.02.024 pmid: 20381137
[17]	Kim E, Goraksha-Hicks P, Li L, et al. Regulation of TORC1 by rag GTPases in nutrient response[J]. Nat Cell Biol, 2008, 10(8): 935-945. doi: 10.1038/ncb1753 pmid: 18604198
[18]	Harrison DA, Perrimon N. Simple and efficient generation of marked clones in Drosophila[J]. Curr Biol, 1993, 3(7): 424-433. pmid: 15335709
[19]	Gonzalez S, Rallis C. The TOR signaling pathway in spatial and temporal control of cell size and growth[J]. Front Cell Dev Biol, 2017, 5: 61. doi: 10.3389/fcell.2017.00061 pmid: 28638821
[20]	Groth AC, Fish M, Nusse R, et al. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31[J]. Genetics, 2004, 166(4): 1775-1782. doi: 10.1534/genetics.166.4.1775 pmid: 15126397
[21]	Thorpe HM, Smith MC. In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family[J]. Proc Natl Acad Sci USA, 1998, 95(10): 5505-5510. doi: 10.1073/pnas.95.10.5505 pmid: 9576912
[22]	Fish MP, Groth AC, Calos MP, et al. Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA and a transgene-containing donor plasmid[J]. Nat Protoc, 2007, 2(10): 2325-2331. doi: 10.1038/nprot.2007.328
[23]	Ni JQ, Markstein M, Binari R, et al. Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster[J]. Nat Methods, 2008, 5(1): 49-51. doi: 10.1038/nmeth1146
[24]	Efeyan A, Schweitzer LD, Bilate AM, et al. RagA, but not RagB, is essential for embryonic development and adult mice[J]. Dev Cell, 2014, 29(3): 321-329. doi: 10.1016/j.devcel.2014.03.017 pmid: 24768164
[25]	Kim J, Kim E. Rag GTPase in amino acid signaling[J]. Amino Acids, 2016, 48(4): 915-928. doi: 10.1007/s00726-016-2171-x pmid: 26781224
[26]	Efeyan A, Zoncu R, Chang S, et al. Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival[J]. Nature, 2013, 493(7434): 679-683. doi: 10.1038/nature11745

引物名称 Primer name	序列 Sequence（5'-3'）
RagA-F	AGTCACTATGGCGGCCGCCATGAAGAAAAAGGTGTTAC
RagA-R	GATGCGGCCTCCACCGCGGCAATGGTACCTTTGGCCATG
M-F	ATCAGAAGCAACACTTGTAAACATAAGGAACGCTC
M-R	TTTACAAGTGTTGCTTCTGATGGCAGCGTGGGATCCG
Q61L-F	ACTGTGGCGGTCTGGAGGGCTTC
Q61L-R	GAAGCCCTCCAGACCGCCACAGT
T16N-F	CCGGAAAGAACAGCATGCGCTC
T16N-R	GAGCGCATGCTGTTCTTTCCGG
RagA-qPCR-F	GCCAGAGCAAGAAGAACC
RagA-qPCR-R	CAATGAAAGCGGCAAAT
rp49-qPCR-F	GCCGCTTCAAGGGACAGT
rp49-qPCR-R	CGATCTCGCCGCAGTAAA

引物名称 Primer name	序列 Sequence（5'-3'）
RagA-F	AGTCACTATGGCGGCCGCCATGAAGAAAAAGGTGTTAC
RagA-R	GATGCGGCCTCCACCGCGGCAATGGTACCTTTGGCCATG
M-F	ATCAGAAGCAACACTTGTAAACATAAGGAACGCTC
M-R	TTTACAAGTGTTGCTTCTGATGGCAGCGTGGGATCCG
Q61L-F	ACTGTGGCGGTCTGGAGGGCTTC
Q61L-R	GAAGCCCTCCAGACCGCCACAGT
T16N-F	CCGGAAAGAACAGCATGCGCTC
T16N-R	GAGCGCATGCTGTTCTTTCCGG
RagA-qPCR-F	GCCAGAGCAAGAAGAACC
RagA-qPCR-R	CAATGAAAGCGGCAAAT
rp49-qPCR-F	GCCGCTTCAAGGGACAGT
rp49-qPCR-R	CGATCTCGCCGCAGTAAA

基因型 Genotype	羽化数 Eclosion number	实际比例 Actual prop- ortions/%	理论比例 Theoretical proportions/%
TM6/UAS-RagA RNAi	897	100	50
Tub-Gal4/UAS-RagA RNAi	0	0	50
总数 Total	897	100	100

基因型 Genotype	羽化数 Eclosion number	实际比例 Actual prop- ortions/%	理论比例 Theoretical proportions/%
TM6/UAS-RagA RNAi	897	100	50
Tub-Gal4/UAS-RagA RNAi	0	0	50
总数 Total	897	100	100

基因型 Genotype	羽化数 Eclosion number	实际比例 Actual pro- portion/%	理论比例 Theoretical proportion/%
UAS-RagA-wt /+; Tub-Gal4/UAS-RagA RNAi	214	14.8	12.5
UAS-RagA-wt /+; Tub-Gal4/MKRS	220	15.3	12.5
UAS-RagA-wt /+; MKRS/TM6	204	14.1	12.5
UAS-RagA-wt /+; UAS-RagA RNAi/TM6	199	13.8	12.5
SM6/+; Tub-Gal4/UAS-RagA RNAi	0	0	12.5
SM6/+; Tub-Gal4/MKRS	192	13.4	12.5
SM6/+; MKRS/TM6	212	14.7	12.5
SM6/+; UAS-RagA RNAi/TM6	201	13.9	12.5
总数 Total	1 442	100	100