Os10g0573900
“OsNMD3”, a highly conserved trans-factor, the 60S ribosomal subunit nuclear export adaptor NMD3. [1]
Contents
Annotated Information
Function
In plants, the process of ribosome biogenesis has remained largely unknown; thus, the exploration of a conserved protein that has been well studied in other organisms is an optimal way to gain insight into ribosomal dynamics. NMD3 is such a protein due to its importance and conservation throughout eukaryotic species [2] [3] [4]. Rice possesses one annotated NMD3 sequence with all representative motifs, including Cx2C repeats at the N-terminus as well as an NLS and NES at the C-terminus. Transient expression assays in rice protoplasts revealed that OsNMD3 shuttles between the nucleus and cytoplasm via CRM1/XPO1, demonstrating its conserved behaviour as a pre-60S nuclear export adaptor, as reported in other species. OsNMD3 was further found to interact with the 60S subunit via OsRPL10Ac1, and it co-sedimented with 60S and 80S ribosome components in ribosome profile analysis.
After being transported from the nucleus, NMD3 must be released from pre-60S particles. This process occurs in the cytoplasm and is an important step toward 60S ribosome maturation [5] [6]. In eukaryotes, the conserved trans-factor NMD3 and eIF6/Tif6 bind to the joining interface with 40S subunit, which blocks premature translation. Release of NMD3 from pre-60S particles can trigger the following events: joining with 40S subunits, processing precursor rRNAs, and forming fully functional ribosomes. Therefore, this step is critical for controlling ribosomal quality for translation [7] [8] [9]. RPL10 and the GTPase LSG1 have been implicated in the release of NMD3 [9]. However, nothing thus far is known about how cytoplasmic maturation of 60S pre-particles occurs in plants. The current work found that OsNMD3ΔNLS was trapped in the cytoplasm. Therefore, truncated OsNMD3 disturbs the cytoplasmic maturation process of 60S particles and affects translational efficiency, which could be further supported by the following findings: (1) both OsNMD3 and OsNMD3ΔNLS bound to OsRPL10Ac1; surplus cytoplasmic OsNMD3ΔNLS may therefore bind to OsRPL10Ac1 and disturb its normal interaction with endogenous OsNMD3; (2) overexpression of OsNMD3 ΔNLS altered the ribosomal structure and interfered with the release of endogenous OsNMD3 from 60S subunits; the presence of OsNMD3ΔNLS in polysomes indicated that cytoplasmic OsNMD3ΔNLS might be loaded onto the ribosome by an unknown mechanism; and (3) transgenic plants showed a reduction in translational efficiency and altered ribosomal structure based on transactivation assays and pharmaceutical treatments with several antibiotics. The protein abundance of CESAs, which participate in cellulose biosynthesis, was significantly low in the OsNMD3ΔNLS transgenic lines, providing solid evidence that OsNMD3ΔNLS decreased the efficiency of protein synthesis. The observation of reduced cellulose content and altered sugar composition was similar to that reported in Arabidopsis plants overexpressing AtNMD3 ΔNES [4]. However, the underlying mechanism differs because the latter was due to the obstruction of AtNMD3 nuclear export. Cell-wall biosynthesis is sensitive to the functionality of nascent ribosomes because these processes are likely to require rapid, abundant protein synthesis. Moreover, RNA sequencing was used to perform a genome-wide examination of the pathways affected by the aberrant 60S particles[10]. The expression level of many factors involved in ribosomal biogenesis was significantly altered in the transgenic plants.
Transgenic Types
To genetically investigate the functions of OsNMD3 in ribosomal dynamics in rice plants, this work employed a dominant negative strategy by overexpression of the GFP-fused wild-type (OsNMD3GFP) and NLS (414–430 aa) truncated forms (OsNMD3ΔNLSGFP) in Nipponbare. The phenotypes of the transgenic plants were examined in the T2 generation. The plants expressing OsNMD3ΔNLSGFP showed dwarfism. From two representative lines (L1 and L2), the plant size of L1 was obviously small compared with that of the wild type. The abnormality was more severe in L2, as the expression level of NMD3ΔNLSGFP was further increased. The L2 plants exhibited pleiotropic phenotypes, including dwarfism and sterility, and they rarely lived to maturity. An observation of the GFP signals in the root cells of these transgenic plants revealed that OsNMD3ΔNLS was localized in the cytoplasm, consistent with the findings obtained with transient expression . However, the plants expressing OsNMD3GFP exhibited a wild-type appearance, although the expression level of OsNMD3 in the transgenic plants was significantly upregulated. The GFP signals in these transgenic plants were found in the cytoplasm and nuclei.
Expression
The presence of NES and NLS motifs indicates that OsNMD3 is nucleocytoplasmically localized. This work therefore fused the green fluorescent protein (GFP) to the C-terminus of full-length OsNMD3 (OsNMD3GFP) and to the C-terminus of truncated OsNMD3 lacking either the NES (OsNMD3ΔNESGFP) or NLS (OsNMD3ΔNLSGFP) and transiently expressed the resulting constructs in rice protoplasts. Fluorescent signals of OsNMD3GFP were observed in both the nucleus and cytoplasm, whereas those of OsNMD3ΔNESGFP and OsNMD3ΔNLSGFP were trapped in the nucleus and cytoplasm, respectively. It has been reported that yeast NMD3 (ScNMD3) acts as a bridge between pre-60S subunits and the nuclear export factor CRM1/XPO1, and leptomycin B (LMB) could block this process [11][12][13]. To investigate whether OsNMD3 shares a similar export pathway with ScNMD3, the current work treated rice protoplasts transiently expressing OsNMD3GFP with LMB and examined the distribution of GFP signals. In contrast to the control cells expressing GFP only, which showed GFP signals in both the nucleus and cytoplasm, the OsNMD3GFP signals were retained in the nucleus after treatment. Taken together, these data indicate that OsNMD3 shuttles between the nucleus and cytoplasm via CRM1/XPO1 and functions as a nuclear export adaptor for 60S ribosomal subunits.
Evolution
NMD3 is one of the highly conserved trans-acting factors and mediates the nuclear export of the 60S ribosomal subunit from yeast to plants [14][15][4]. Through a BlastP search of the rice genome, the current work identified only one annotated NMD3 sequence with an open reading frame located at the locus LOC_Os10g42320 (Rice Genome Annotation Project, http://rice.plantbiology.msu.edu/) or Os10g0573900 (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/). An unrooted phylogenetic tree was further constructed including the NMD3 from 18 representative species of bacteria, animals, and plants using the neighbour-joining method. OsNMD3 was clustered with sorghum NMD3 (SbNMD3) into a monophyletic clade that arose before the divergence of monocot and dicot phyla. Despite different evolutionary scenarios, OsNMD3 is conserved among the examined species. Within the length of 523 amino acids, OsNMD3 has four cysteine repeat motifs (Cx2C) at the N-terminus with an NLS (414–430 aa) and a leucine-rich NES (494–503 aa) at the C-terminus. These motifs are highly conserved among all eukaryotic cells, indicating that OsNMD3 may play roles in 60S ribosome dynamics that are similar to those reported in other species.
Labs working on this gene
1. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
References
- ↑ 1.0 1.1 1.2 1.3 Shi Y, Liu X, Li R, Gao Y, Xu Z, Zhang B, Zhou Y. Retention of OsNMD3 in the cytoplasm disturbs protein synthesis efficiency and affects plant development in rice. Journal of Experimental Botany, 2014, 65(12): 3055-3069.
- ↑ Johnson AW, Lund E, Dahlberg J. 2002. Nuclear export of ribosomal subunits.Trends in Biochemical Science 27, 580–585.
- ↑ Zemp I, Kutay U. 2007. Nuclear export and cytoplasmic maturation of ribosomal subunits. FEBS Letter 581, 2783–2793.
- ↑ 4.0 4.1 4.2 Chen MQ, Zhang AH, Zhang Q, et al. 2012. Arabidopsis NMD3 is required for nuclear export of 60S ribosomal subunits and affects secondary cell wall thickening. PLoS ONE 7, e35904.
- ↑ Panse VG, Johnson AW. 2010. Maturation of eukaryotic ribosomes: acquisition of functionality. Trends in Biochemical Science 35, 260–266.
- ↑ Karbstein K. 2013. Quality control mechanisms during ribosome maturation. Trends in Cell Biology 23, 242–250.
- ↑ Gartmann M, Blau M, Armache JP, Mielke T, Topf M, Beckmann R. 2010. Mechanism of eIF6-mediated inhibition of ribosomal subunit joining. Journal of Biological Chemistry 285, 14848–14851.
- ↑ Sengupta J, Bussiere C, Pallesen J, West M, Johnson AW, Frank J. 2010. Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit. Journal of Cell Biology 189, 1079–1086.
- ↑ 9.0 9.1 Klinge S, Voigts-Hoffmann F, Leibundgut M, Arpagaus S, Ban N. 2011. Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6. Science 334, 941–948.
- ↑ Hedges J, West M, Johnson AW. 2005. Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p. EMBO Journal 24, 567–579.
- ↑ Ho JH, Kallstrom G, Johnson AW. 2000. Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit. Journal of Cell Biology 151, 1057–1066.
- ↑ Gadal O, Strauss D, Kessl J, Trumpower B, Tollervey D, Hurt E. 2001. Nuclear export of 60S ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p. Molecular Cell Biology 21, 3405–3415.
- ↑ Kressler D, Hurt E, Bassler J. 2010. Driving ribosome assembly. Biochimica et Biophysica Acta 1803, 673–683.
- ↑ Ho JH, Johnson AW. 1999. NMD3 encodes an essential cytoplasmic protein required for stable 60S ribosomal subunits in Saccharomyces cerevisiae . Molecular Cell Biology 19, 2389–2399.
- ↑ Thomas F, Kutay U. 2003. Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway. Journal of Cell Science 116, 2409–2419.
