Exon-intron boundary inhibits mA deposition, enabling mA distribution hallmark, longer mRNA half-life and flexible protein coding.

Regional bias of N-methyladenosine (mA) mRNA modification avoiding splice site region, calls for an open hypothesis whether exon-intron boundary could affect mA deposition. By deep learning modeling, we find that exon-intron boundary represses a proportion (12% to 34%) of mA deposition at adjacent exons (~100 nt to splice site). Experiments validate that mA signal increases once the host gene does not undergo pre-mRNA splicing to produce the same mRNA.

Deep learning modeling mA deposition reveals the importance of downstream cis-element sequences.

The N-methyladenosine (mA) modification is deposited to nascent transcripts on chromatin, but its site-specificity mechanism is mostly unknown. Here we model the mA deposition to pre-mRNA by iM6A (intelligent mA), a deep learning method, demonstrating that the site-specific mA methylation is primarily determined by the flanking nucleotide sequences. iM6A accurately models the mA deposition (AUROC = 0.

Saturation mutagenesis reveals manifold determinants of exon definition.

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells.

Pre-mRNA processing includes methylation of adenosine residues that are retained in mRNA exons and the fallacy of "RNA epigenetics".

By using a cell fraction technique that separates chromatin-associated nascent RNA, newly completed nucleoplasmic mRNA and cytoplasmic mRNA, we have shown in a previous study that residues in exons are methylated (mA) in nascent pre-mRNA and remain methylated in the same exonic residues in nucleoplasmic and cytoplasmic mRNA. Thus, there is no evidence of a substantial degree of demethylation in mRNA exons that would correspond to so-called "epigenetic" demethylation. The turnover rate of mRNA molecules is faster, depending on mA content in HeLa cell mRNA, suggesting that specification of mRNA stability may be the major role of mA exon modification.

mA mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover.

Understanding the biologic role of -methyladenosine (mA) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and mA in exons but very rarely in introns. The mA methylation is essentially completed upon the release of mRNA into the nucleoplasm.

A majority of m6A residues are in the last exons, allowing the potential for 3' UTR regulation.

We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m(6)A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3'-most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m(6)A and >40% of all m(6)A in mRNA are present in 3' untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m(6)A sites around stop codons.

Quantitative evaluation of all hexamers as exonic splicing elements.

We describe a comprehensive quantitative measure of the splicing impact of a complete set of RNA 6-mer sequences by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells. The results allowed the assignment of a relative splicing strength score to each mutant molecule.

Context-dependent splicing regulation: exon definition, co-occurring motif pairs and tissue specificity.

Splicing is a crucial process in gene expression in higher organisms because: 1) most vertebrate genes contain introns; and 2) alternative splicing is primarily responsible for increasing proteomic complexity and functional diversity. Intron definition, the coordination across an intron, is a mandatory step in the splicing process. However, exon definition, the coordination across an exon, is also thought to be required for the splicing of most vertebrate exons.

Intronic motif pairs cooperate across exons to promote pre-mRNA splicing.

Background: A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions.

Splicing of designer exons reveals unexpected complexity in pre-mRNA splicing.

Pre-messengerRNA (mRNA) splicing requires the accurate recognition of splice sites by the cellular RNA processing machinery. In addition to sequences that comprise the branchpoint and the 3' and 5' splice sites, the cellular splicing machinery relies on additional information in the form of exonic and intronic splicing enhancer and silencer sequences. The high abundance of these motifs makes it difficult to investigate their effects using standard genetic perturbations, since their disruption often leads to the formation of yet new elements.

Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density.

Drought is one of the most important environmental constraints limiting plant growth and agricultural productivity. To understand the underlying mechanism of drought tolerance and to identify genes for improving this important trait, we conducted a gain-of-function genetic screen for improved drought tolerance in Arabidopsis thaliana. One mutant with improved drought tolerance was isolated and designated as enhanced drought tolerance1.

Positive selection acting on splicing motifs reflects compensatory evolution.

We have used comparative genomics to characterize the evolutionary behavior of predicted splicing regulatory motifs. Using base substitution rates in intronic regions as a calibrator for neutral change, we found a strong avoidance of synonymous substitutions that disrupt predicted exonic splicing enhancers or create predicted exonic splicing silencers. These results attest to the functionality of the hexameric motif set used and suggest that they are subject to purifying selection.