Interspecies transfer of syntenic RAMOSA1 orthologs and promoter cis sequences impacts maize inflorescence architecture

Grass inflorescences support floral structures that each bear a single grain, where variation in branch architecture directly impacts yield. The maize RAMOSA1 (ZmRA1) transcription factor acts as a key regulator of inflorescence development by imposing branch meristem determinacy. Here, we show RA1 transcripts accumulate in boundary domains adjacent to spikelet meristems in Sorghum bicolor (Sb) and Setaria viridis (Sv) inflorescences similar as in the developing maize tassel and ear. To evaluate functional conservation of syntenic RA1 orthologs and promoter cis sequences in maize, sorghum and setaria, we utilized interspecies gene transfer and assayed genetic complementation in a common inbred background by quantifying recovery of normal branching in highly ramified ra1-R mutants. A ZmRA1 transgene that includes endogenous upstream and downstream flanking sequences recovered normal tassel and ear branching in ra1-R. Interspecies expression of two transgene variants of the SbRA1 locus, modeled as the entire endogenous tandem duplication or just the non-frameshifted downstream copy, complemented ra1-R branching defects and induced novel fasciation and branch patterns. The SvRA1 locus lacks conserved, upstream noncoding cis sequences found in maize and sorghum; interspecies expression of an SvRA1 transgene did not or only partially recovered normal inflorescence forms. Driving expression of the SvRA1 coding region by the ZmRA1 upstream region, however, recovered normal inflorescence morphology in ra1-R. These data leveraging interspecies gene transfer suggest that cis-encoded temporal regulation of RA1 expression is a key factor in modulating branch meristem determinacy that ultimately impacts grass inflorescence architecture.

PlantRNA 2.0 : an updated database dedicated to tRNAs of photosynthetic eukaryotes

PlantRNA (http://plantrna.ibmp.cnrs.fr/) is a comprehensive database of transfer RNA (tRNA) gene sequences retrieved from fully annotated nuclear, plastidial and mitochondrial genomes of photosynthetic organisms. In the first release (PlantRNA 1.0), tRNA genes from 11 organisms were annotated. In this second version, the annotation was implemented to 48 photosynthetic species covering the whole phylogenetic tree of photosynthetic organisms, from the most basal group of Archeplastida, the glaucophyte Cyanophora paradoxa, to various land plants. Transfer RNA genes from lower photosynthetic organisms such as streptophyte algae or lycophytes as well as extremophile photosynthetic species such as Eutrema parvulum were incorporated in the database. As a whole, circa 35 000 tRNA genes were accurately annotated. In the frame of the tRNA genes annotation from the genome of the Rhodophyte Chondrus crispus, putative unconventional splicing sites in the D- or T- regions of tRNA molecules were experimentally determined to strengthen the quality of the database. As for PlantRNA 1.0, comprehensive biological information including flanking sequences, A and B box sequences, region of transcription initiation and poly(T) transcription termination stretches, tRNA intron sequences and tRNA mitochondrial import are included.

Toxicity of internalized polyalanine to cells depends on aggregation

In polyalanine (PA) diseases, the disease-causing transcription factors contain an expansion of alanine repeats. While aggregated proteins that are responsible for the pathogenesis of neurodegenerative disorders show cell-to-cell propagation and thereby exert toxic effects on the recipient cells, whether this is also the case with expanded PA has not been studied. It is also not known whether the internalized PA is toxic to recipient cells based on the degree of aggregation. In this study, we therefore prepared different degrees of aggregation of a peptide having 13 alanine repeats without flanking sequences of PA disease-causative proteins (13A). The aggregated 13A was spontaneously taken up by neuron-like cultured cells. Functionally, strong aggregates but not weak aggregates displayed a deficit in neuron-like differentiation in vitro. Moreover, the injection of strong but not weak 13A aggregates into the ventricle of mice during the neonatal stage led to enhanced spontaneous motor activity later in life. Thus, PA in the extracellular space has the potential to enter adjacent cells, and may exert toxicity depending on the degree of aggregation.

InMut-finder: a software tool for insertion identification in mutagenesis using Nanopore long reads

Background Biological mutagens (such as transposon) with sequences inserted, play a crucial role to link observed phenotype and genotype in reverse genetic studies. For this reason, accurate and efficient software tools for identifying insertion sites based on the analysis of sequencing reads are desired. Results We developed a bioinformatics tool, a Finder, to identify genome-wide Insertions in Mutagenesis (named as “InMut-Finder”), based on target sequences and flanking sequences from long reads, such as Oxford Nanopore Sequencing. InMut-Finder succeeded in identify > 100 insertion sites in Medicago truncatula and soybean mutants based on sequencing reads of whole-genome DNA or enriched insertion-site DNA fragments. Insertion sites discovered by InMut-Finder were validated by PCR experiments. Conclusion InMut-Finder is a comprehensive and powerful tool for automated insertion detection from Nanopore long reads. The simplicity, efficiency, and flexibility of InMut-Finder make it a valuable tool for functional genomics and forward and reverse genetics. InMut-Finder was implemented with Perl, R, and Shell scripts, which are independent of the OS. The source code and instructions can be accessed at https://github.com/jsg200830/InMut-Finder.

Clinical feature-related single-base substitution sequence signatures identified with an unsupervised machine learning approach

Background Mutation processes leave different signatures in genes. For single-base substitutions, previous studies have suggested that mutation signatures are not only reflected in mutation bases but also in neighboring bases. However, because of the lack of a method to identify features of long sequences next to mutation bases, the understanding of how flanking sequences influence mutation signatures is limited. Methods We constructed a long short-term memory-self organizing map (LSTM-SOM) unsupervised neural network. By extracting mutated sequence features via LSTM and clustering similar features with the SOM, single-base substitutions in The Cancer Genome Atlas database were clustered according to both their mutation site and flanking sequences. The relationship between mutation sequence signatures and clinical features was then analyzed. Finally, we clustered patients into different classes according to the composition of the mutation sequence signatures by the K-means method and then studied the differences in clinical features and survival between classes. Results Ten classes of mutant sequence signatures (mutation blots, MBs) were obtained from 2,141,527 single-base substitutions via LSTM-SOM machine learning approach. Different features in mutation bases and flanking sequences were revealed among MBs. MBs reflect both the site and pathological features of cancers. MBs were related to clinical features, including age, sex, and cancer stage. The class of an MB in a given gene was associated with survival. Finally, patients were clustered into 7 classes according to the MB composition. Significant differences in survival and clinical features were observed among different patient classes. Conclusions We provided a method for analyzing the characteristics of mutant sequences. Result of this study showed that flanking sequences, together with mutation bases, shape the signatures of SBSs. MBs were shown related to clinical features and survival of cancer patients. Composition of MBs is a feasible predictive factor of clinical prognosis. Further study of the mechanism of MBs related to cancer characteristics is suggested.

Differential Regulation of Human Surfactant Protein A Genes, SFTPA1 and SFTPA2, and Their Corresponding Variants

The human SFTPA1 and SFTPA2 genes encode the surfactant protein A1 (SP-A1) and SP-A2, respectively, and they have been identified with significant genetic and epigenetic variability including sequence, deletion/insertions, and splice variants. The surfactant proteins, SP-A1 and SP-A2, and their corresponding variants play important roles in several processes of innate immunity as well in surfactant-related functions as reviewed elsewhere [1]. The levels of SP-A have been shown to differ among individuals both under baseline conditions and in response to various agents or disease states. Moreover, a number of agents have been shown to differentially regulate SFTPA1 and SFTPA2 transcripts. The focus in this review is on the differential regulation of SFTPA1 and SFTPA2 with primary focus on the role of 5′ and 3′ untranslated regions (UTRs) and flanking sequences on this differential regulation as well molecules that may mediate the differential regulation.

Complete Chloroplast Genome Sequence of Erigeron breviscapus and Characterization of Chloroplast Regulatory Elements

Erigeron breviscapus is a famous medicinal plant. However, the limited chloroplast genome information of E. breviscapus, especially for the chloroplast DNA sequence resources, has hindered the study of E. breviscapus chloroplast genome transformation. Here, the complete chloroplast (cp) genome of E. breviscapus was reported. This genome was 152,164bp in length, included 37.2% GC content and was structurally arranged into two 24,699bp inverted repeats (IRs) and two single-copy areas. The sizes of the large single-copy region and the small single-copy region were 84,657 and 18,109bp, respectively. The E. breviscapus cp genome consisted of 127 coding genes, including 83 protein coding genes, 36 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. For those genes, 95 genes were single copy genes and 16 genes were duplicated in two inverted regions with seven tRNAs, four rRNAs, and five protein coding genes. Then, genomic DNA of E. breviscapus was used as a template, and the endogenous 5' and 3' flanking sequences of the trnI gene and trnA gene were selected as homologous recombinant fragments in vector construction and cloned through PCR. The endogenous 5' flanking sequences of the psbA gene and rrn16S gene, the endogenous 3' flanking sequences of the psbA gene, rbcL gene, and rps16 gene and one sequence element from the psbN-psbH chloroplast operon were cloned, and certain chloroplast regulatory elements were identified. Two homologous recombination fragments and all of these elements were constructed into the cloning vector pBluescript SK (+) to yield a series of chloroplast expression vectors, which harbored the reporter gene EGFP and the selectable marker aadA gene. After identification, the chloroplast expression vectors were transformed into Escherichia coli and the function of predicted regulatory elements was confirmed by a spectinomycin resistance test and fluorescence intensity measurement. The results indicated that aadA gene and EGFP gene were efficiently expressed under the regulation of predicted regulatory elements and the chloroplast expression vector had been successfully constructed, thereby providing a solid foundation for establishing subsequent E. breviscapus chloroplast transformation system and genetic improvement of E. breviscapus.

The different activities of RNA G-quadruplex structures are controlled by flanking sequences

The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.

Fine-mapping of nuclear compartments using ultra-deep Hi-C shows that active promoter and enhancer elements localize in the active A compartment even when adjacent sequences do not

Megabase-scale intervals of active, gene-rich and inactive, gene-poor chromatin are known to segregate, forming the A and B compartments. Fine mapping of the contents of these A and B compartments has been hitherto impossible, owing to the extraordinary sequencing depths required to distinguish between the long-range contact patterns of individual loci, and to the computational complexity of the associated calculations. Here, we generate the largest published in situ Hi-C map to date, spanning 33 billion contacts. We also develop a computational method, dubbed PCA of Sparse, Super Massive Matrices (POSSUMM), that is capable of efficiently calculating eigenvectors for sparse matrices with millions of rows and columns. Applying POSSUMM to our Hi-C dataset makes it possible to assign loci to the A and B compartment at 500 bp resolution. We find that loci frequently alternate between compartments as one moves along the contour of the genome, such that the median compartment interval is only 12.5 kb long. Contrary to the findings in coarse-resolution compartment profiles, we find that individual genes are not uniformly positioned in either the A compartment or the B compartment. Instead, essentially all (95%) active gene promoters localize in the A compartment, but the likelihood of localizing in the A compartment declines along the body of active genes, such that the transcriptional termini of long genes (>60 kb) tend to localize in the B compartment. Similarly, essentially all active enhancers elements (95%) localize in the A compartment, even when the flanking sequences are comprised entirely of inactive chromatin and localize in the B compartment. These results are consistent with a model in which DNA-bound regulatory complexes give rise to phase separation at the scale of individual DNA elements.

Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon

Background Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown. Results We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3ʹ-end. Consequently, the secondary structures were changed, the RNA–protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3ʹ-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs. Conclusions Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study. Graphic Abstract