Selective recruitment of mRNAs and miRNAs to the translational machinery

MAURICIO REYNOSO, FLAVIO BLANCO, JULIA BAILEY-SERRES, MARTÍN CRESPI, MARIA EUGENIA ZANETTI

Cold Spring Harbor New York

Simposio; 77 th CSHL Symposium: The biology of plants; 2012

Cold Spring Harbor Laboratory

Translation of mRNAs plays a crucial role in the regulation of gene expression during development or in response to environmental stimuli (1). The symbiotic interaction between legume and rhizobia involves two highly coordinated processes, the nodule organogenesis and the bacterial infection. The interaction is initiated by the perception of the bacteria, which suppresses plant defence responses, infects the roots tissue and colonizes the developing nodule primordium (2). Several studies have characterized the steady-state levels of mRNAs at different stages of nitrogen fixing symbiosis. However, abundance of an mRNA does not necessarily reflect its translation status. In addition, microRNAs (miRNAs) have emerged as major regulators of mRNA translation or stability. Hence, we characterized changes in the association of mRNAs and miRNAs to polyrribosome (polysomes) in roots of the model legume Medicago truncatula during the symbiotic interaction with Sinorhizobium meliloti. Purification of polysomes was achieved by immunoprecipitation (IP) using root tissue expressing a FLAG-tagged ribosomal protein (RP) L18 (3, 4). Quantitative comparison between S. melilotiMedicago truncatula during the symbiotic interaction with Sinorhizobium meliloti. Purification of polysomes was achieved by immunoprecipitation (IP) using root tissue expressing a FLAG-tagged ribosomal protein (RP) L18 (3, 4). Quantitative comparison between S. melilotiSinorhizobium meliloti. Purification of polysomes was achieved by immunoprecipitation (IP) using root tissue expressing a FLAG-tagged ribosomal protein (RP) L18 (3, 4). Quantitative comparison between S. melilotiS. meliloti inoculated and non inoculated roots revealed differential translational status for genes of the nodulation signalling pathway. Three translational categories were defined: 1) transcripts up-regulated at translational level, which includes NFP and CRE1 receptors, the GRAS family transcription factors NSP1 and NSP2, and the HAP2-1 and HAP5b subunits of the CCAAT binding factor; 2) translational down-regulated transcripts, represented by the cation channel DMI1 and 3) transcripts not regulated at translational level, including the transcription factors NIN and ERN, the early nodulin ENOD40, the LYK3 receptor and the calcium calmodulin dependent kinase DMI3. Quantitative analysis of sRNAs associated with the immunopurified polysome detected several mature microRNAs (miRNAs), notably miR169d, which targets the 3`UTR of the HAP2-1 transcript (5). Association of this miRNA with polysomes significantly decreased upon inoculation with S. meliloti in correlation with increased levels of HAP2-1 protein. Current experiments are being conducted to explore translational changes at genome scale combining the IP of polysomes with RNA sequencing technology. 1) Bailey Serres et al (2009). Trends in Plant Sci 14, 443. 2) Oldroyd et al (2011). Annu Rev Genet 45, 119. 3) Zanetti et al (2005) Plant Phys 138, 624 4) Mustroph et al, (2009) PNAS USA 106, 18843 5) Devers et al, 2011. Plant Phys 156, 1990. This work was supported by ANPCyT (PICT 2007-00095) and by an International cooperation program of CONICET of Argentina and NSF of USA.S. meliloti in correlation with increased levels of HAP2-1 protein. Current experiments are being conducted to explore translational changes at genome scale combining the IP of polysomes with RNA sequencing technology. 1) Bailey Serres et al (2009). Trends in Plant Sci 14, 443. 2) Oldroyd et al (2011). Annu Rev Genet 45, 119. 3) Zanetti et al (2005) Plant Phys 138, 624 4) Mustroph et al, (2009) PNAS USA 106, 18843 5) Devers et al, 2011. Plant Phys 156, 1990. This work was supported by ANPCyT (PICT 2007-00095) and by an International cooperation program of CONICET of Argentina and NSF of USA.Trends in Plant Sci 14, 443. 2) Oldroyd et al (2011). Annu Rev Genet 45, 119. 3) Zanetti et al (2005) Plant Phys 138, 624 4) Mustroph et al, (2009) PNAS USA 106, 18843 5) Devers et al, 2011. Plant Phys 156, 1990. This work was supported by ANPCyT (PICT 2007-00095) and by an International cooperation program of CONICET of Argentina and NSF of USA.45, 119. 3) Zanetti et al (2005) Plant Phys 138, 624 4) Mustroph et al, (2009) PNAS USA 106, 18843 5) Devers et al, 2011. Plant Phys 156, 1990. This work was supported by ANPCyT (PICT 2007-00095) and by an International cooperation program of CONICET of Argentina and NSF of USA.138, 624 4) Mustroph et al, (2009) PNAS USA 106, 18843 5) Devers et al, 2011. Plant Phys 156, 1990. This work was supported by ANPCyT (PICT 2007-00095) and by an International cooperation program of CONICET of Argentina and NSF of USA.106, 18843 5) Devers et al, 2011. Plant Phys 156, 1990. This work was supported by ANPCyT (PICT 2007-00095) and by an International cooperation program of CONICET of Argentina and NSF of USA.156, 1990. This work was supported by ANPCyT (PICT 2007-00095) and by an International cooperation program of CONICET of Argentina and NSF of USA.