Research Article: An artificial miRNA system reveals that relative contribution of translational inhibition to miRNA-mediated regulation depends on environmental and developmental factors in Arabidopsis thaliana

Date Published: February 16, 2018

Publisher: Public Library of Science

Author(s): Patrick von Born, Marti Bernardo-Faura, Ignacio Rubio-Somoza, Sebastien Pfeffer.


Development and fitness of any organism rely on properly controlled gene expression. This is especially true for plants, as their development is determined by both internal and external cues. MicroRNAs (miRNAs) are embedded in the genetic cascades that integrate and translate those cues into developmental programs. miRNAs negatively regulate their target genes mainly post-transcriptionally through two co-existing mechanisms; mRNA cleavage and translational inhibition. Despite our increasing knowledge about the genetic and biochemical processes involved in those concurrent mechanisms, little is known about their relative contributions to the overall miRNA-mediated regulation. Here we show that co-existence of cleavage and translational inhibition is dependent on growth temperature and developmental stage. We found that efficiency of an artificial miRNA-mediated (amiRNA) gene silencing declines with age during vegetative development in a temperature-dependent manner. That decline is mainly due to a reduction on the contribution from translational inhibition. Both, temperature and developmental stage were also found to affect mature amiRNA accumulation and the expression patterns of the core players involved in miRNA biogenesis and action. Therefore, that suggests that each miRNA family specifically regulates their respective targets, while temperature and growth might influence the performance of miRNA-dependent regulation in a more general way.

Partial Text

Control of gene expression is paramount for any organism in order to exist and transit through different developmental stages as well as to interrelate with their surroundings during their life cycle. All layers controlling gene expression are tightly regulated, from chromatin state to protein post-translational modifications, including mRNA stability. Small RNAs (sRNAs) have emerged in the last decades as central elements embedded in those regulatory layers. sRNAs come in several flavors depending on the source of RNA used for their biogenesis [1]. MicroRNAs (miRNAs) are a special class of sRNAs that mainly regulate the expression of their targets post-transcriptionally. miRNA-dependent regulation has evolved independently in at least six eukaryotic lineages, including land plants [2]. Most of the current knowledge about plant miRNA biogenesis, action and function comes from studies in the model organism Arabidopsis thaliana. Primary miRNA transcripts (pri-miRNA) arise from the RNA polymerase II-dependent expression of independent transcriptional units. Their expression pattern is under the control of specific regulatory sequences as is the case for protein coding genes [3]. Pri-miRNAs are processed by the microprocessor complex in mature miRNA duplexes ranging from 19 to 24 nt at the dicing bodies within the nuclei in a two-step enzymatic reaction [4]. Proteins from the DICER family, mainly DICER-LIKE1 (DCL1; [5]) are the core components of the microprocessor complex and are assisted by accessory proteins such as HYPONASTIC LEAVES1 (HYL1; [6]) or DOUBLE-RNA BINDING PROTEIN 2 (DRB2; [7]), SERRATE (SE; [8]) and C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1; [9]). The resulting mature miRNA duplexes are subsequently protected from degradation through HUA-ENHANCER 1 (HEN1)-mediated methylation [5]. Next, HASTY (HST; [10]) is thought to participate in the transport of the stabilized miRNA duplexes to the cytoplasm where they are loaded into the RNA-Induced Silencing Complex (RISC). Proteins from the ARGONAUTE (AGO) family are the main executive components of the RISC complex. The Arabidopsis genome has 10 AGO genes of which AGO1 [11] and AGO10 are considered the main players in post-transcriptional miRNA-mediated gene silencing [12]. Once loaded into the RISC, one of the two duplex strands is degraded while the remaining one serves to scan the cytoplasm seeking for highly complementary mRNAs. miRNAs control the expression of their targets both by mRNA-target cleavage and translational inhibition [12]. Beyond their co-existence, knowledge about the overall contribution of both mechanisms in plants is scarce and suggests that it might be cell-type specific [13]. Noteworthy, the implications of both regulatory mechanisms on miRNA-mediated regulation are critical for its dynamics. While mRNA cleavage and degradation is a non-reversible process, it is thought that translational inhibition might be reverted allowing for a rapid expression of its repressed targets [14].

Our findings show that plant miRNA performance (accumulation, efficiency and co-existence of target cleavage and translational inhibition) is influenced by both development and environment. Our results support that the expression of several central players in miRNA performance also depends on development and temperature in which plants are grown.




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