High quality genome assembly of the amitochondriate eukaryote Monocercomonoides exilis

Monocercomonoides exilis is considered the first known eukaryote to completely lack mitochondria. This conclusion is based primarily on a genomic and transcriptomic study which failed to identify any mitochondrial hallmark proteins. However, the available genome assembly has limited contiguity and around 1.5 % of the genome sequence is represented by unknown bases. To improve the contiguity, we re-sequenced the genome and transcriptome of M. exilis using Oxford Nanopore Technology (ONT). The resulting draft genome is assembled in 101 contigs with an N50 value of 1.38 Mbp, almost 20 times higher than the previously published assembly. Using a newly generated ONT transcriptome, we further improve the gene prediction and add high quality untranslated region (UTR) annotations, in which we identify two putative polyadenylation signals present in the 3′UTR regions and characterise the Kozak sequence in the 5′UTR regions. All these improvements are reflected by higher BUSCO genome completeness values. Regardless of an overall more complete genome assembly without missing bases and a better gene prediction, we still failed to identify any mitochondrial hallmark genes, thus further supporting the hypothesis on the absence of mitochondrion.

Analysis of SINE Families B2, Dip, and Ves with Special Reference to Polyadenylation Signals and Transcription Terminators

Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3′-terminus of many mammalian SINEs has a polyadenylation signal (AATAAA), pol III transcription terminator, and A-rich tail. The RNAs of such SINEs can be polyadenylated, which is unique for pol III transcripts. Here, B2 (mice and related rodents), Dip (jerboas), and Ves (vespertilionid bats) SINE families were thoroughly studied. They were divided into subfamilies reliably distinguished by relatively long indels. The age of SINE subfamilies can be estimated, which allows us to reconstruct their evolution. The youngest and most active variants of SINE subfamilies were given special attention. The shortest pol III transcription terminators are TCTTT (B2), TATTT (Ves and Dip), and the rarer TTTT. The last nucleotide of the terminator is often not transcribed; accordingly, the truncated terminator of its descendant becomes nonfunctional. The incidence of complete transcription of the TCTTT terminator is twice higher compared to TTTT and thus functional terminators are more likely preserved in daughter SINE copies. Young copies have long poly(A) tails; however, they gradually shorten in host generations. Unexpectedly, the tail shortening below A10 increases the incidence of terminator elongation by Ts thus restoring its efficiency. This process can be critical for the maintenance of SINE activity in the genome.

The RNA-Binding Protein HuD Regulates Alternative Splicing and Alternative Polyadenylation in the Mouse Neocortex

The neuronal Hu/ELAV-like proteins HuB, HuC and HuD are a class of RNA-binding proteins that are crucial for proper development and maintenance of the nervous system. These proteins bind to AU-rich elements (AREs) in the untranslated regions (3′-UTRs) of target mRNAs regulating mRNA stability, transport and translation. In addition to these cytoplasmic functions, Hu proteins have been implicated in alternative splicing and alternative polyadenylation in the nucleus. The purpose of this study was to identify transcriptome-wide effects of HuD deletion on both of these nuclear events using RNA sequencing data obtained from the neocortex of Elavl4–/– (HuD KO) mice. HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of proximal polyadenylation signals (PAS), resulting in shorter 3′-UTRs. None of these genes overlapped with those showing alternative splicing events. Overall, HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.

ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing

ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member,DrosophilaElav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3’ UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets ofDrosophilaare known, ectopic expression of each of the three family members (Elav, Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants ofelav/fne, but notelavalone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation ofDrosophilaELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3’ UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification inDrosophilaand mammalian neurons.

The Evolution of G-quadruplex Structure in mRNA Untranslated Region

The RNA G-quadruplex (rG4) is a kind of non-canonical high-order secondary structure with important biological functions and is enriched in untranslated regions (UTRs) of protein-coding genes. However, how rG4 structures evolve is largely unknown. Here, we systematically investigated the evolution of RNA sequences around UTR rG4 structures in 5 eukaryotic organisms. We found universal selection on UTR sequences, which facilitated rG4 formation in all the organisms that we analyzed. While G-rich sequences were preferred in the rG4 structural region, C-rich sequences were selectively not preferred. The selective pressure acting on rG4 structures in the UTRs of genes with higher G content was significantly smaller. Furthermore, we found that rG4 structures experienced smaller evolutionary selection near the translation initiation region in the 5′ UTR, near the polyadenylation signals in the 3′ UTR, and in regions flanking the miRNA targets in the 3′ UTR. These results suggest universal selection for rG4 formation in the UTRs of eukaryotic genomes and the selection may be related to the biological functions of rG4s.

SINE Retrotransposons Import Polyadenylation Signals to 3’UTRs in Dog (Canis familiaris)

BackgroundMessenger RNA 3’ untranslated regions (3’UTRs) control many aspects of gene expression and determine where the transcript will terminate. The polyadenylation signal (PAS) AAUAAA is a key regulator of transcript termination and this hexamer, or a similar sequence, is very frequently found within 30 bp of 3’UTR ends. Short interspersed element (SINE) retrotransposons are found throughout genomes in high copy number. When inserted into genes they can disrupt expression, alter splicing, or cause nuclear retention of mRNAs. The genomes of the domestic dog and other carnivores carry hundreds of thousands Can-SINEs, a tRNA-related SINE with transcription termination potential. Because of this we asked whether Can-SINEs may help terminate transcript in some dog genes.ResultsDog 3’UTRs have several peaks of AATAAA PAS frequency within 40 bp of the 3’UTR end, including four bp-interval peaks at 28, 32, and 36 bp from the end. The periodicity is partly explained by TAAA(n) repeats within Can-SINE AT-rich tails. While density of antisense-oriented Can-SINEs in 3’UTRs is fairly constant with distances from 3’end, sense-oriented Can-SINEs are common at the 3’end but nearly absent farther upstream. There are nine Can-SINE sub-types in the dog genome and the consensus sequence sense strands (head to tail) all carry at least three PASs while antisense strands usually have none. We annotated all repeat-masked Can-SINE copies in the Boxer reference genome and found that the young SINEC_Cf type has a mode of 15 bp for target site duplications (TSDs). We find that all Can-SINE types favor integration at TSDs beginning with A(4). The count of AATAAA PASs differs significantly between sense and antisense-oriented retrotransposons in transcripts. Can-SINEs near 3’UTR ends are very likely to carry AATAAA on the mRNA sense strand while those farther upstream are not. We also identified loci where Can-SINE insertion has truncated or altered a dog 3’UTR compared to the human ortholog.ConclusionDog Can-SINE activity has imported AATAAA PASs into gene transcripts and led to alteration of 3’UTRs. AATAAA sequences are selectively removed from Can-SINEs in introns and upstream 3’UTR regions but are retained at the far downstream end of 3’UTRs, which we infer reflects their role as termination sequences for these transcripts.

Animal-APAdb: a comprehensive animal alternative polyadenylation database

Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism that recognizes different polyadenylation signals on transcripts, resulting in transcripts with different lengths of 3′ untranslated regions and thereby influencing a series of biological processes. Recent studies have highlighted the important roles of APA in human. However, APA profiles in other animals have not been fully recognized, and there is no database that provides comprehensive APA information for other animals except human. Here, by using the RNA sequencing data collected from public databases, we systematically characterized the APA profiles in 9244 samples of 18 species. In total, we identified 342 952 APA events with a median of 17 020 per species using the DaPars2 algorithm, and 315 691 APA events with a median of 17 953 per species using the QAPA algorithm in these 18 species, respectively. In addition, we predicted the polyadenylation sites (PAS) and motifs near PAS of these species. We further developed Animal-APAdb, a user-friendly database (http://gong_lab.hzau.edu.cn/Animal-APAdb/) for data searching, browsing and downloading. With comprehensive information of APA events in different tissues of different species, Animal-APAdb may greatly facilitate the exploration of animal APA patterns and novel mechanisms, gene expression regulation and APA evolution across tissues and species.

Transcription terminator-mediated enhancement in transgene expression in maize: preponderance of the AUGAAU motif overlapping with poly(A) signals

The selection of transcription terminators (TTs) for pairing with high expressing constitutive promoters in chimeric constructs is crucial to deliver optimal transgene expression in plants. In this study, the use of the native combinations of four polyubiquitin gene promoters and corresponding TTs resulted in up to >3-fold increase in transgene expression in maize. Of the eight polyubiquitin promoter and TT regulatory elements utilized, seven were novel and identified from the polyubiquitin genes of Brachypodium distachyon, Setaria italica, and Zea mays. Furthermore, gene expression driven by the Cassava mosaic virus promoter was studied by pairing the promoter with distinct TTs derived from the high expressing genes of Arabidopsis. Of the three TTs studied, the polyubiquitin10 gene TT produced the highest transgene expression in maize. Polyadenylation patterns and mRNA abundance from eight distinct TTs were analyzed using 3’-RACE and next-generation sequencing. The results exhibited one to three unique polyadenylation sites in the TTs. The poly(A) site patterns for the StPinII TT were consistent when the same TT was deployed in chimeric constructs irrespective of the reporter gene and promoter used. Distal to the poly(A) sites, putative polyadenylation signals were identified in the near-upstream regions of the TTs based on previously reported mutagenesis and bioinformatics studies in rice and Arabidopsis. The putative polyadenylation signals were 9 to 11 nucleotides in length. Six of the eight TTs contained the putative polyadenylation signals that were overlaps of either canonical AAUAAA or AAUAAA-like polyadenylation signals and AUGAAU, a top-ranking-hexamer of rice and Arabidopsis gene near-upstream regions. Three of the polyubiquitin gene TTs contained the identical 9-nucleotide overlap, AUGAAUAAG, underscoring the functional significance of such overlaps in mRNA 3’ end processing. In addition to identifying new combinations of regulatory elements for high constitutive trait gene expression in maize, this study demonstrated the importance of TTs for optimizing gene expression in plants. Learning from this study could be applied to other dicotyledonous and monocotyledonous plant species for transgene expression.