Heat Stress-induced Alternative Splicing Provides A Novel Mechanism For The Regulation Of MicroRNA Processing In Plants

Environmental stress factors seriously influence plant growth and development.Extensive agricultural losses are attributed to temperature, often in combination with droughtor other stresses. A number of genes were proved to be involved in plant responses to variousabiotic stresses. The regulation of gene expression is mainly controlled at the transcriptionallevel. Previous studies have indicated that AS (Alternative Splicing) is common in plants andcontributes to both transcriptome and proteome diversity. Several reports have shown thatvarious environmental stresses influence alternative splicing of pre-mRNAs, including somestress response genes.Recently, microRNAs (miRNAs), a class of~22nt noncoding RNAs, have emerged asregulators of gene expression through post-transcriptional degradation or translationalrepression through base pairing to target mRNAs. The discovery of large numbers of miRNAsand their diverse functions in both plants and animals has led to widespread agreement abouttheir importance. Several recent studies have shown that a number of stress-regulated genesencoding important transcription factors are targets of miRNAs. However, increasingevidence points to the important relationship between miRNAs and environmental stressresponses, but the regulatory mechanisms in plants are poorly understood. To our knowledge,no direct evidence of how stress-related AS regulates miRNA expression has been provided inprevious research.Here, a novel example is provided that stress-related AS could be a regulatorymechanism modulating the expression of intronic miRNAs. The findings of this study extendthe current view about the regulatory mechanism of miRNAs. The main results andconclusions presented in this thesis are as follows:(1) To understand the regulatory mechanism of the temperature affecting miR400expression, we performed various transcript analyses. Sequence analysis showed that miR400derived from the first intron of At1g32583in the5′UTR. Both the tissue pattern of GUSstaining and qRT-RCR analysis revealed that miR400was encoded in the intron of At1g32583 and was transcribed as a single unit in various tissues. Taken together, intronic miR400isco-transcripted with the hostgene in various tissues of Arabidopsis.(2) To investigate the relationship between miR400expression and cold stress inArabidopsis, we performed qRT-PCR and northern blot analysis. Increased levels of MIR400and host gene transcripts were apparent after8h of exposure to4℃and continued to increasewith prolonged (24h) exposure. The level of mature miR400was also upregulated by coldstress, suggesting that the accumulation of both MIR400transcripts and mature miR400wasdue to transcriptional induction in response to cold stress. Analysis of GUS activity andhistochemical GUS staining showed that cold Stress induced miR400and its hostgeneexpression mainly by increasing transcriptional activity.To further examine the miR400response to heat stress, we performed qRT-PCR andnorthern blot analysis using seedlings exposed to a temperature of37℃for12h. Surprisingly,expression of MIR400and its host gene displayed opposite responses to heat stress. Heatstress suppresses mature miR400expression but upregulates MIR400transcripts.(3) Intriguingly, we found pre-mRNAs underwent an alternative splicing event in the firstintron of the host gene downstream of MIR400transcripts under heat treatment. A100bpexcised fragment from the3′-end of the first intron was detected. Thus, we suggest that thedeletion of100bp fragments interfered with the splicing of the remaining region of the firstintron containing miR400, resulting in the accumulation of MIR400transcripts.To further understand how AS influences intronic miR400processing, we generated fourintronic miR400constructs to directly test the effects of the100bp excised fragments in theAS event. The results showed that AS-mediated regulation resulted in decreased miR400levels by holding the206bp retained intron with the primary miR400hairpin in the5′UTR ofthe host genes. We conclude that the100bp excised fragment appearing in the heat-inducedAS event is essential for the processing of mature miR400in Arabidopsis. Together,alternative splicing is the major factor for inhibiting mature miR400expression under heatstress.(4) To further understand how AS suppresses miR400expression, we performed adetailed sequence analysis of the first intron in the host gene. The results showed that the secondary structure of pri-miR400was not interrupted after the heat-induced AS event, whichcould be folded to enable processing of the miRNA when the holding intron is spliced out ofthe pre-mRNA. A typical branch point likely mediates normal processing of miR400. Wefound that the100bp fragment contained the original branch point from the3′-end of theexcised intron, whereas the remaining206bp intron was retained, indicating that the splicingsignals in the retained sequences were not sufficiently clear to be recognized as intronic.Thus,the recognition of the pre-mRNA branch point and associated sequences is essential fornormal processing of miR400.(5) To further characterize the biological function of miR400in heat stress, we generatedtransgenic Arabidopsis plants overexpressing miR400under control of the constitutive CaMV35S promoter. No phenotype differences were observed between the transgenic and wild typeplants under normal growth conditions. However, under heat stress,35S::MIR400seeds had alower germination percentage and germinated slower compared with the wild type seeds. Andthe less hypocotyl elongation of35S::MIR400seedlings indicated a defective in acquiredthermotolerance. Moreover, heat treatment also reduced the root growth of35S::MIR400seedlings. Together, these results revealed that overexpression of MIR400made the plantsmore sensitive to heat stress, suggesting that miR400might play a role in regulating theresponse to environmental stresses in plants.To date,17intronic miRNAs have been discovered in Arabidopsis. Sequence analysis ofthe introns containing miRNAs showed that eight miRNAs have potential alternative splicingisoforms, suggesting that the generation of other intronic miRNAs might be affected by ASevents triggered under specific conditions in plants (as in the case of miR400). AS acts as aregulatory mechanism for miRNA expression in response to environmental cues and wepropose that the insights gained from the characterization of miR400will be useful forstudying many other miRNAs present in eukaryotic genomes. Future investigations areneeded to determine whether AS might be involved in other intronic miRNA cropping inplants.