Bioinformatics Analysis Of The Regulatory Role Of Alternative Splicing Regulating In Mineral Nutrient Homeostasis In Rice(Oryza Sativa)

Plants have evolved flexible and complex regulatory systems that allow them to adapt to differences and changes in nutrient availability in the soil.Because the nutrient-use efficiency directly affects the yield and quality of crops,the molecular multi-level regulation mechanism for the uptake,transport,distribution and utilization of plant mineral elements needs to be deeply investigated.Previous studies mainly focus on transcription factor(TFs)centered transcriptional regulation,and alternative splicing(AS)studies in plant functional genomics are not extensively carried out and the biological significance of AS in higher plants has been even underestimated.AS of genes not only enriches protein diversity,but also fine-tune the regulation of gene expression as a post-transcriptional regulation mechanism.It plays important roles in a wide range of biological processes,including responses to plant abiotic stress,plant defense against pathogens,cell differentiation fate determination,growth and development,circadian rhythms regulation and flower timing.In order to demonstrate that AS plays a role in regulating mineral nutrient homeostasis in rice,we conduct a genome-wide comparative alternative splicing analysis study under mineral nutrient deficiency and between OsSCL57 mutant and WT using RNA-Seq.To profile the model plant rice transcriptome and AS in response to mineral deficiency,2-week-old rice seedlings were grown under control(replete)or Fe-,Zn-,Cu-,or Mn-deficient(depleted)growth conditions for 10 days,the micronutrient deficiency status of rice seedlings was confirmed by phenotype observation,concentration measurement of trace metals and expression analysis of several metal homeostasis genes,then RNA-Seq was carried out on rice roots under control and micronutrient deficient conditions using pair-end reads of Illumina Hiseq platform.Two RNA-Seq datasets from polyA-Enrich and rRNA-Zero libraries per sample were obtained.Although the two libraries provide high consistent gene expression quantification and biologically relevant differential expressed gene signatures,the quantification and differential splicing analsis of alternative splicing event revealed that rRNA-Zero sequencing method detected more reads from not completely spliced transcripts in the cell nucleus,resulting in a significantly higher intron retention ratio.To analyze rRNA-Zero RNA-Seq dataset,we develop a statistical procedure with the assumption that the per-base coverage depth of individual intronic positions is fitting poisson distribution with rate parameter ? estimated by average per-base coverage depth.For each intron,the average per-base coverage depth of each intron and the significant P-value based on the Poisson distribution was calculated,the specificity and sensitivity analysis for comparing assembled transcripts of rRNA-Zero library after filtering not fully spliced isoforms by different P-value cutoffs with assembled transcripts of polyA-Enrich library was performed and the optimal P-value cutoff was selected.The obvious advantage of the post-assembly transcript filtering method is that it can be widely used in the assembled transcripts from accurate,comprehensive and efficient assembly tools,such as StringTie,which are more suitable for the assembly of polyA-Enrich sequencing reads.Only two assembly tools,Cufflinks and CLASS2 also consider intronic noise,which is produced by pre-mRNA unspliced transcripts and affect the accuracy of alternative splicing isoform assembly,but it is integrated in the transcription assembly process.Intron retention is the most common AS event in plants,the method decreased the high rate of false-positive intron retention events predicted by the pre-mRNA splicing intermediate products.We also obtained publicly available root rRNA-Zero RNA-seq dataset derived from a previous Pi-starvation time course study(1 hours,6 hours,3 days,7 days,21 days,21 days+1 hours,21 days+6 hours and 22 days).Genome-wide comparative analysis of alternative splicing event and gene was performed using whole transcriptome RNA sequencing data of the same rRNA-Zero library and sequencing platform under micronutrient and Pi deficiency.In the study,we used our in-house bioinformatics pipeline to analyze RNA-Seq datasets under mineral nutrient deficiency.124,590 transcripts and 50,678 genes in total were identified.A total of 53,471 AS events were predicted using rMATS software.These AS events accounted for?37.7%of the total 12,180 intron-containing genes in the rice genome.Intron retention(IR)events were the predominant AS event type,accounting for?37.7%,followed by alternative 3' splice site(A3SS)(?16.1%),alternative 5' splice site(A5SS)(?10.6%),exon skipping(ES)(?5.5%),and mutually exclusive exons(MXE)(?0.08%).Differentially expressed genes(DEGs)and differentially alternatively spliced genes(DASGs)under each nutrient deficiency were detected.The less than 10%overlap between DEGs and DASGs under each nutrient deficiency revealed that alternative splicing regulatory mechanism is relatively independant of transcriptional regulation.The overlap analysis of DASGs under mineral nutrient deficiency conditions revealed that the targets of AS were highly specific to each nutrient.To characterise global changes in alternative splicing under nutrient deficiency,AS gene ratio,defined as the total number of AS genes divided by the total number of intron-containing genes was defined for each condition.It was observed that the global AS gene ratio among Pi-deplete(-P)samples was significantly higher than Pi-replete(+P)samples in the root.Intron retention event of one sulfate transporter gene(Os03g0195800)would induce a premature termination codon(PTC),resulting in a C-terminal truncation protein.The relative expression level of the isoform encoding the short protein significantly increased under-Fe.Alternative splicing of this sulfate transporter gene would potentially affect sulfate homeostasis under-Fe.Furthermore,the second intron retention event of OsSPX1 was induced under-P,and reduce the efficiency of mature mRNA export from the nucleus to the cytoplasm.OsSPX1 protein can interact with positive regulatory transcription factor OsPHR2.AS of OsSPX1 tightly regulated the expression level of protein isoform,and plays a negative regulatory role in the phosphate signaling network.To validate the reliablity of our statistical method for AS event identification,four differential AS events under Pi deficiecy were selected and confirmed using quantitative reverse transcription-PCR(RT-PCR).The RT-PCR results showed the same trend as the RNA-seq data.Members of the SR(Serine/Arginine-rich)protein gene family play a key role in plant abiotic stress responses through the regulation of AS.The Pfam domain enrichment analysis results for high-frequancy AS genes show that the RNA binding protein gene family with RNA recognition motif(RRM)is more likely to undergo AS.The transcription levels of 22 SR gene were not strongly regulated by mineral deficiency in roots.14 SR genes had one or more significantly differentially alternatively spliced events during micronutrient and Pi deficiency.We predict the impact of differential splicing events on transcript structure and protein function for four SR genes(OsRS33,OsSCL25,OsRSZ21a and OsSR40).Some AS events regulated transcript levels by regulating unproductive splicing and translation mechanism(RUST)through the coupling of alternative splicing and nonsense-mediated mRNA decay(NMD).Other AS events resulted in a truncated protein containing the RRM domain,which interferes with the wild-type protein's function,or change the 3' UTR length,which may affect the stability or translation efficiency of its mRNA.These alternative splicing regulation mechaisms of SR genes participate in plant responses to nutrient stress by finely regulating SR protein homeostasis.Previously,we have screened a rice OsSCL57 gene mutant of which inorganic Pi content significantly accumulates in roots ammong sr mutants.OsSCL57 was one SR gene of the plant specific SCL protein subfamily.PolyA-Enrich RNA sequencing was performed in 12-day rice roots of osscl57 mutants using WT as a control.By observing read coverage plot,T-DNA was inserted into the eighth intron of the OsSCL57 gene,resulting in incomplete C-terminal SR function domain.Two phosphate transporters OsPT2 and OsPT8 were significantly up-regulated in the mutant.3,977 differentially alternative spliced event and 2,506 associated genes were identified between WT and mutant.The first intron inclusive ratio of phosphate signaling negative regulator OsSPX4 significantly increases in the mutant,resulting in the reduction of protein-coding transcript expression level.Furthermore,the fifth intron retention event of the positive regulator OsARF16 leads to the increase of the protein-coding transcript expression level.These above results suggest that OsSCL57 fine-tune the expression level of keys genes involved in phosphate uptake,transport and signaling transduction through alternative splicing regulation mechanism.The AS regulatory mechanism contributes to the phenotype of phosphorus accumulation in the root of mutant.Comparative alternative splicing analysis under micronutrient and Pi deficiency and osscl57 mutant analysis using RNA-Seq revealed that AS plays a critical role in regulating mineral nutrient homestasis in rice.We develop a short-read based alternative splicing analysis pipeline,which provides an important reference for large-scale AS study of other plants.Our findings from alternative splicing analysis of Pi-related genes in osscl57 mutant have potential applications for generating crops with increased P use efficiency.