Phospho-proteomics identifies a critical role of ATF2 in pseudorabies virus replication.

Pseudorabies virus (PRV), an etiological agent of pseudorabies in livestock, has negatively affected the porcine industry all over the world. Epithelial cells are reported as the first site of PRV infection. However, the role of host proteins and its related signaling pathways in PRV replication is largely unclear. In this study, we performed a quantitative phosphoproteomics screening on PRV-infected porcine kidney (PK-15) epithelial cells. Totally 5723 phosphopeptides, corresponding to 2180 proteins, were obtained, and the phosphorylated states of 810 proteins were significantly different in PRV-infected cells compared with mock-infected cells (P â€‹< â€‹0.05). GO and KEGG analysis revealed that these differentially expressed phosphorylated proteins were predominantly related to RNA transport and MAPK signaling pathways. Further functional studies of NF-κB, transcription activator factor-2 (ATF2), MAX and SOS genes in MAPK signaling pathway were analyzed using RNA interference (RNAi) knockdown. It showed that only ATF2-knockdown reduces both PRV titer and viral genome copy number. JNK pathway inhibition and CRISPR/Cas9 gene knockout showed that ATF2 was required for the effective replication of PRV, especially during the biogenesis of viral genome DNA. Subsequently, by overexpression of the ATF2 gene and point mutation of the amino acid positions 69/71 of ATF2, it was further demonstrated that the phosphorylation of ATF2 promoted PRV replication. These findings suggest that ATF2 may provide potential therapeutic target for inhibiting PRV infection.

Knockout of glutathione peroxidase 5 down-regulates the piRNAs in the caput epididymidis of aged mice.

The mammalian epididymis not only plays a fundamental role in the maturation of spermatozoa, but also provides protection against various stressors. The foremost among these is the threat posed by oxidative stress, which arises from an imbalance in reactive oxygen species and can elicit damage to cellular lipids, proteins, and nucleic acids. In mice, the risk of oxidative damage to spermatozoa is mitigated through the expression and secretion of glutathione peroxidase 5 (GPX5) as a major luminal scavenger in the proximal caput epididymidal segment. Accordingly, the loss of GPX5-mediated protection leads to impaired DNA integrity in the spermatozoa of aged Gpx5-/- mice. To explore the underlying mechanism, we have conducted transcriptomic analysis of caput epididymidal epithelial cells from aged (13 months old) Gpx5-/- mice. This analysis revealed the dysregulation of several thousand epididymal mRNA transcripts, including the downregulation of a subgroup of piRNA pathway genes, in aged Gpx5-/- mice. In agreement with these findings, we also observed the loss of piRNAs, which potentially bind to the P-element-induced wimpy testis (PIWI)-like proteins PIWIL1 and PIWIL2. The absence of these piRNAs was correlated with the elevated mRNA levels of their putative gene targets in the caput epididymidis of Gpx5-/- mice. Importantly, the oxidative stress response genes tend to have more targeting piRNAs, and many of them were among the top increased genes upon the loss of GPX5. Taken together, our findings suggest the existence of a previously uncharacterized somatic piRNA pathway in the mammalian epididymis and its possible involvement in the aging and oxidative stress-mediated responses.

[Assessing abundance and specificity of different types of sgRNA targeting miRNA precursors].

CRISPR/Cas technology enables efficient and specific editing the genome. Since different bacterial sources or artificially modified Cas9, as well as Cpf1 and other nucleases, recognize different PAMs (protospacer adjacent motifs), different gene editing nucleases may use different types of sgRNAs (small guide RNA). MicroRNAs (miRNAs) are a class of regulatory small non-coding RNAs. To determine whether specific targets for sgRNAs in miRNA precursor exit, the abundance and specificity of 11 different types of sgRNA targeting 28 645 miRNA precursors were analyzed in the present study using the CRISPR-offinder, a bioinformatics software developed in our own laboratory. The CRISPR/Cas9 lentivirus technology was used to target the miR-302/367 cluster in a porcine cell line, and its knockout efficiency for the miRNA target was evaluated. The results show that there are about 8 different types of sgRNAs that can target individual miRNA precursors. By assessing the off-target effect, only 18.2% of the sgRNAs showed high specificity for targeting the porcine miRNA precursors. Lastly, using the miR-302/367 cluster target as an example, we showed that the CRISPR/Cas9 lentivirus technology was 40% efficient in successfully establishing correct knockout of the target miRNA in the porcine cell line. This present study provides an important resource for the use of CRISPR/Cas technology to target miRNAs for knockout studies.

Comparative microRNAome analysis of the testis and ovary of the Chinese giant salamander.

MicroRNAs (miRNAs) are 18-24 nucleotides non-coding RNAs that regulate gene expression by post-transcriptional suppression of mRNA. The Chinese giant salamander (CGS, Andrias davidianus), which is an endangered species, has become one of the important models of animal evolution; however, no miRNA studies on this species have been conducted. In this study, two small RNA libraries of CGS ovary and testis were constructed using deep sequencing technology. A bioinformatics pipeline was developed to distinguish miRNA sequences from other classes of small RNAs represented in the sequencing data. We found that many miRNAs and other small RNAs such as piRNA and tsRNA were abundant in CGS tissue. A total of 757 and 756 unique miRNAs were annotated as miRNA candidates in the ovary and testis respectively. We identified 145 miRNAs in CGS ovary and 155 miRNAs in CGS testis that were homologous to those in Xenopus laevis ovary and testis respectively. Forty-five miRNAs were more highly expressed in ovary than in testis and 21 miRNAs were more highly expressed in testis than in ovary. The expression profiles of the selected miRNAs (miR-451, miR-10c, miR-101, miR-202, miR-7a and miR-499) had their own different roles in other eight tissues and different development stages of testis and ovary, suggesting that these miRNAs play vital regulatory roles in sexual differentiation, gametogenesis and development in CGS. To our knowledge, this is the first study to reveal miRNA profiles that are related to male and female CGS gonads and provide insights into sex differences in miRNA expression in CGS.

[sgRNA design for the CRISPR/Cas9 system and evaluation of its off-target effects].

The third generation of CRISPR/Cas9-mediated genome editing technology has been successfully applied to genome modification of various species including animals, plants and microorganisms. How to improve the efficiency of CRISPR/Cas9 genome editing and reduce its off-target effects has been extensively explored in this field. Using sgRNA (Small guide RNA) with high efficiency and specificity is one of the critical factors for successful genome editing. Several software have been developed for sgRNA design and/or off-target evaluation, which have advantages and disadvantages respectively. In this review, we summarize characters of 16 kinds online and standalone software for sgRNA design and/or off-target evaluation and conduct a comparative analysis of these different kinds of software through developing 38 evaluation indexes. We also summarize 11 experimental approaches for testing genome editing efficiency and off-target effects as well as how to screen highly efficient and specific sgRNA.

Development of a graphical user interface for sgRNAcas9 and its application.

The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, off-target effects of the Cas9 nuclease activity is a recurrent concern of the CRISPR system. Thus, designing sgRNA (single guide RNA) with minimal off-target effects is very important. sgRNAcas9 is a software package, which can be used to design sgRNA and to evaluate potential off-target cleavage sites. In this study, a graphical user interface for sgRNAcas9 was developed using the Java programming language. In addition, off-target effect for sgRNAs was evaluated according to mismatched number and "seed sequence" specification. Moreover, sgRNAcas9 software was used to design 34 124 sgRNAs, which can target 4691 microRNA (miRNA) precursors from human, mouse, rat, pig, and chicken. In particular, the off-target effect of a sgRNA targeting to human miR-206 precursor was analyzed, and the on/off-target activity of this sgRNA was validated by T7E1 assay in vitro. Taken together, these data showed that the interface can simplify the usage of the sgRNAcas9 program, which can be used to design sgRNAs for the majority of miRNA precursors. We also found that the GC% of those sgRNAs ranged from 40% to 60%. In summary, the sgRNAcas9 software can be easily used to design sgRNA with minimal off-target effects for any species. The software can be downloaded from BiooTools website (http://www.biootools.com/).

Discovery of porcine microRNAs in multiple tissues by a Solexa deep sequencing approach.

The domestic pig (Sus scrofa) is an important economic animal for meat production and as a suitable model organism for comparative genomics and biomedical studies. In an effort to gain further identification of miRNAs in the pig, we have applied the Illumina Solexa sequencing technology to carry out an in-depth analysis of the miRNA transcriptome in a pool of equal amounts of RNA from 16 different porcine tissues. From this data set, we identified 437 conserved and 86 candidate novel miRNA/miRNA* in the pig, corresponding to 329 miRNA genes. Compared with all the reported porcine miRNAs, the result showed that 112 conserved and 61 candidate novel porcine miRNA were first reported in this study. Further analysis revealed extensive sequence variations (isomiRs) of porcine miRNAs, including terminal isomiRs at both the 5' and 3' ends and nucleotide variants. Read counts of individual porcine miRNA spanned from a few reads to approximately 405541 reads, confirming the different level of expression of porcine miRNAs. Subsequently, the tissue expression patterns of 8 miRNAs were characterized by Northern blotting. The results showed that miR-145, miR-423-5p, miR-320, miR-26a, and miR-191 are ubiquitously expressed in diverse tissues, while miR-92, miR-200a, and miR-375 were selectively enriched and expressed in special tissues. Meanwhile, the expression of 8 novel porcine-specific miRNAs was validated by stem-loop RT-PCR, and one of these was detected by Northern blotting. Using the porcine miRNA array designed according to our Solexa results, 123 miRNAs were detected expression in porcine liver tissues. A total of 58 miRNAs showed differential expression between the Tongcheng (a Chinese indigenous fatty breed) and Large White pig breeds (a lean type pig). Taken together, our results add new information to existing data on porcine miRNAs and should be useful for investigating the biological functions of miRNAs in pig and other species.