RNA-sequencing, commonly referred to as RNA-seq, is the most recently developed method for the analysis of transcriptomes. It uses high-throughput next-generation sequencing technologies and has revolutionized our understanding of the complexity and dynamics of whole transcriptomes.In this chapter, we recall the key developments in transcriptome analysis and dissect the different steps of the general workflow that can be run by users to design and perform a mRNA-seq experiment as well as to process mRNA-seq data obtained by the Illumina technology. The chapter proposes guidelines for completing a mRNA-seq study properly and makes available recommendations for best practices based on recent literature and on the latest developments in technology and algorithms. We also remark the large number of choices available (especially for bioinformatic data analysis) in front of which the scientist may be in trouble.In the last part of the chapter we discuss the new frontiers of single-cell RNA-seq and isoform sequencing by long read technology.

Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3\'-untranslated region (3\'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3\'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3\'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3\'-UTRs to achieve the faithful transition of germ cells to meiosis.

Granule-bound starch synthase (GBSS), a product of the waxy gene in rice (Oryza sativa L.), is necessary for the synthesis of amylose in the endosperm. In an extended pedigree of 89 rice cultivars, we have previously shown that all cultivars with more than 18% amylose had the sequence AGGTATA at the leader intron 5\' splice site, while all cultivars with a lower proportion of amylose had the sequence AGTTATA. This single-nucleotide polymorphism reduces the efficiency of GBSS pre-mRNA processing. It also results in alternate splicing at multiple sites, some of which have non-consensus sequences. Here we demonstrate that this same G-to-T polymorphism is also associated with differential sensitivity to temperature during the period of grain development. Cultivars with the sequence AGTTATA have a substantial increase in accumulation of mature GBSS transcripts at 18 degrees C compared to 25 or 32 degrees C. The selection of leader intron 5\' splice sites is also affected by temperature in these cultivars. A 5\' splice site -93 upstream from that used in high-amylose varieties predominates at 18 degrees C. At higher temperatures there is increased utilization of a 5\' splice site at -I and a non-consensus site at +1. Potential implications of differential 5\' splice site selection and associated differences in 3\' splice site selection on transcript stability and translational efficiency are discussed.

The promoter regions of most plastid transcription units have been reported to consist of prokaryotic -10 and -35-like consensus sequences. However, a few promoters with no homology to the consensus elements have also been characterized. A novel class of non-consensus plastid promoters--designated as non consensus type II (NC-II) promoters--that effect low-level constitutive expression of respective genes in photosynthetic as well as non-photosynthetic plastids is described in this paper. The abundance of NC-II promoter-derived transcripts remains unaltered even when light-grown seedlings are shifted to the dark. In contrast, transcripts from -10 and -35-like elements containing consensus type (CT) promoters accumulate to high levels in chloroplasts as compared with non-photosynthetic plastids of roots and cultured cells. Moreover, accumulation of these transcripts is greatly affected by light. The inhibition of plastid protein synthesis has no apparent effect on the abundance of the NC-II transcripts whereas levels of CT transcripts are greatly reduced. In vivo tagetitoxin (a plastid transcription inhibitor) treatment also reduces the levels of CT transcripts with no apparent inhibitory effect on the accumulation of NC-II transcripts. The accumulation of transcripts from both classes of promoters, however, is reduced when cytoplasmic protein synthesis is inhibited by in vivo treatment with cycloheximide. The results are suggestive of the possible existence of at least two distinctive systems for the synthesis and/or maintenance of plastid transcripts which differentiate between two classes of transcripts in a promoter-type specific manner.

Intron lariat formation between the 5\' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3\' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3\' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.