Genome-wide profiling of untranslated regions by paired-end ditag sequencing reveals unexpected transcriptome complexity in yeast

Abstract

The identification of structural and functional elements encoded in a genome is a challenging task. Although the transcriptome of budding yeast has been extensively analyzed, the boundaries and untranslated regions of yeast genes remain elusive. To address this least-explored field of yeast genomics, we performed a transcript profiling analysis through paired-end ditag (PET) approach coupled with deep sequencing. With 562,133 PET sequences we accurately defined the boundaries and untranslated regions of 3,409 ORFs, suggesting many yeast genes have multiple transcription start sites (TSSs). We also identified 85 previously uncharacterized transcripts either in intergenic regions or from the opposite strand of reported genomic features. Furthermore, our data revealed the extensive 3' end heterogeneity of yeast genes and identified a novel putative motif for polyadenylation. Our results indicate the yeast transcriptome is more complex than expected. This study would serve as an invaluable resource for elucidating the regulation and evolution of yeast genes.

Cell-to-cell heterogeneity of phosphate gene expression in yeast is controlled by alternative transcription, 14-3-3 and Spl2

“…1) hybridizes to RNA of approximately 600 nucleotides, slightly larger that found observed before (approx. 500 nucleotides [52]). The amount of RNA hybridizing to this probe was strongly increased after deletion of box P, both in BY4741 and in rrp6Δ.…”

MALAT1 long ncRNA promotes gastric cancer metastasis by suppressing PCDH10

“…Briefly, isolated cell nuclei were used for RNA immunoprecipitation with EZH2-specific antibody and the enriched RNAs were reverse transcribed using a random primer flanked with a 7 nucleotide (nt) barcode sequence to track strand information. The second strand cDNA synthesis was performed using the adaptor ligation strategy we reported recently [ 15 ]. The resulting RIP libraries were amplified and subjected to Illumina sequencing (Figure 1A ).…”

“…The 1st strand cDNA was synthesized with random primer flanked by a 7 nt sequence (Adaptor 1, Supplementary Table 5 ) to track strand information. The 2nd strand DNA was generated by the strategy we previously reported [ 15 ] with N6 adaptor (Adaptor 2, Supplementary Table 5 ). The resulting DNA libraries were amplified with Phusion ® High-Fidelity DNA Polymerase (NEB) as follows: 30 sec at 98°C, followed by a 4 cycles of (10 sec at 98°C, 10 sec at 66°C, 30 sec at 72°C) and 14–20 cycles of (10 sec at 98°C, 10 sec at 69°C, 30 sec at 72°C), additional extension for 10 min at 72°C and then hold at 10°C.…”

CLASS2: accurate and efficient splice variant annotation from RNA-seq reads

“…Better methods are needed to characterize the various types of artifacts that confound classes of variations, such as alternative polyadenylation or alternative promoter usage and retained introns. These can entail implementing sequence models of binding sites of regulatory proteins ( 31 , 32 ), or incorporating other types of evidence including CAGE tags, DNase-seq or FAIRE-seq signals, paired-end diTags (PET-seq) ( 33 ) and polyA-seq ( 34 ) sequences, where available. Also needed are complete reference data sets on genes or systems that can help evaluate the performance in an unbiased way, or at the very least better simulation models.…”