Identification and Comparative Analysis of the Tegillarca granosa Haemocytes MicroRNA Transcriptome in Response to Cd Using a Deep Sequencing Approach

Background

MicroRNAs (miRNAs) are endogenous non-coding small RNAs (sRNAs) that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. To identify miRNAs regulated by heavy metal stress, we constructed two sRNA libraries for the blood clam Tegillarca granosa: one for organisms exposed to toxic levels of cadmium (Cd) and one for a control group.

Results

Sequencing of the two libraries and subsequent analysis revealed 215 conserved and 39 new miRNAs. Most of the new miRNAs in T. granosa were up- or down-regulated in response to Cd exposure. There were significant differences in expression between the Cd and control groups for 16 miRNAs. Of these, five miRNAs were significantly up-regulated and 11 were significantly down-regulated in the Cd stress library. Potential targets were predicted for the 16 differential miRNAs in pre-miRNAs identified according to sequence homology. Some of the predicted miRNA targets are associated with regulation of the response to stress induced by heavy metals. Five differentially expressed miRNAs (Tgr-nmiR-8, Tgr-nmiR-21, Tgr-miR-2a, Tgr-miR-10a-5p, and Tgr-miR-184b) were validated by qRT-PCR.

Conclusion

Our study is the first large-scale identification of miRNAs in T. granosa haemocytes. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in the responses of this species to environmental heavy metal stresses.     

Sex specific expression and distribution of small RNAs in papaya

Background

Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored.

Results

We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot.

Conclusion

By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Y^h chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-20) contains supplementary material, which is available to authorized users.     

Expression and localization of microRNAs in perinatal rat pancreas: role of miR-21 in regulation of cholesterol metabolism

Objective

To investigate the expression of pancreatic microRNAs (miRNAs) during the period of perinatal beta-cell expansion and maturation in rats, determine the localization of these miRNAs and perform a pathway analysis with predicted target mRNAs expressed in perinatal pancreas.

Research Design and Methods

RNA was extracted from whole pancreas at embryonic day 20 (E20), on the day of birth (P0) and two days after birth (P2) and hybridized to miRNA microarrays. Differentially expressed miRNAs were verified by northern blotting and their pancreatic localization determined by in situ hybridization. Pathway analysis was done using regulated sets of mRNAs predicted as targets of the miRNAs. Possible target genes were tested using reporter-gene analysis in INS-1E cells.

Results

Nine miRNAs were differentially expressed perinatally, seven were confirmed to be regulated at the level of the mature miRNA. The localization studies showed endocrine localization of six of these miRNAs (miR-21, -23a, -29a, -125b-5p, -376b-3p and -451), and all were expressed in exocrine cells at one time point at least. Pathways involving metabolic processes, terpenoid and sterol metabolism were selectively affected by concomitant regulation by miRNAs and mRNAs, and Srebf1 was validated as a target of miR-21.

Conclusions

The findings suggest that miRNAs are involved in the functional maturation of pancreatic exocrine and endocrine tissue following birth. Pathway analysis of target genes identify changes in sterol metabolism around birth as being selectively affected by differential miRNA expression during this period.     

Genome-wide identification of vegetative phase transition-associated microRNAs and target predictions using degradome sequencing in Malus hupehensis

Background

A long juvenile period between germination and flowering is a common characteristic among fruit trees, including Malus hupehensis (Pamp.) Rehd., which is an apple rootstock widely used in China. microRNAs (miRNAs) play an important role in the regulation of phase transition and reproductive growth processes.

Results

M. hupehensis RNA libraries, one adult and one juvenile phase, were constructed using tree leaves and underwent high-throughput sequencing. We identified 42 known miRNA families and 172 novel miRNAs. We also identified 127 targets for 25 known miRNA families and 168 targets for 35 unique novel miRNAs using degradome sequencing. The identified miRNA targets were categorized into 58 biological processes, and the 123 targets of known miRNAs were associated with phase transition processes. The KEGG analysis revealed that these targets were involved in starch and sucrose metabolism, and plant hormone signal transduction. Expression profiling of miRNAs and their targets indicated multiple regulatory functions in the phase transition. The higher expression level of mdm-miR156 and lower expression level of mdm-miR172 in the juvenile phase leaves implied that these two small miRNAs regulated the phase transition. mdm-miR160 and miRNA393, which regulate genes involved in auxin signal transduction, could also be involved in controlling this process. The identification of known and novel miRNAs and their targets provides new information on this regulatory process in M. hupehensis, which will contribute to the understanding of miRNA functions during growth, phase transition and reproduction in woody fruit trees.

Conclusions

The combination of sRNA and degradome sequencing can be used to better illustrate the profiling of hormone-regulated miRNAs and miRNA targets involving complex regulatory networks, which will contribute to the understanding of miRNA functions during growth, phase transition and reproductive growth in perennial woody fruit trees.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1125) contains supplementary material, which is available to authorized users.     

Integrated miRNA-mRNA Analysis Revealing the Potential Roles of miRNAs in Chordomas

Introduction

Emerging evidence suggests that microRNAs (miRNAs) are crucially involved in tumorigenesis and that paired expression profiles of miRNAs and mRNAs can be used to identify functional miRNA-target relationships with high precision. However, no studies have applied integrated analysis to miRNA and mRNA profiles in chordomas. The purpose of this study was to provide insights into the pathogenesis of chordomas by using this integrated analysis method.

Methods

Differentially expressed miRNAs and mRNAs of chordomas (n = 3) and notochord tissues (n = 3) were analyzed by using microarrays with hierarchical clustering analysis. Subsequently, the target genes of the differentially expressed miRNAs were predicted and overlapped with the differentially expressed mRNAs. Then, GO and pathway analyses were performed for the intersecting genes.

Results

The microarray analysis indicated that 33 miRNAs and 2,791 mRNAs were significantly dysregulated between the two groups. Among the 2,791 mRNAs, 911 overlapped with putative miRNA target genes. A pathway analysis showed that the MAPK pathway was consistently enriched in the chordoma tissue and that miR-149-3p, miR-663a, miR-1908, miR-2861 and miR-3185 likely play important roles in the regulation of MAPK pathways. Furthermore, the Notch signaling pathway and the loss of the calcification or ossification capacity of the notochord may also be involved in chordoma pathogenesis.

Conclusion

This study provides an integrated dataset of the miRNA and mRNA profiles in chordomas, and the results demonstrate that not only the MAPK pathway and its related miRNAs but also the Notch pathway may be involved in chordoma development. The occurrence of chordoma may be associated with dysfunctional calcification or ossification of the notochord.     

Identification of Wolbachia-responsive microRNAs in the two-spotted spider mite,Tetranychus urticae

Background

The two-spotted spider mite, Tetranychus urticae, is infected with Wolbachia, which have the ability to manipulate host reproduction and fitness. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes such as development, reproduction and host-pathogen interactions. Although miRNA was observed to involve in Wolbachia-host interactions in the other insect systems, its roles have not been fully deciphered in the two-spotted spider mite.

Results

Small RNA libraries of infected and uninfected T. urticae for both sexes (in total four libraries) were constructed. By integrating the mRNA data originated from the same samples, the target genes of the differentially expressed miRNAs were predicted. Then, GO and pathway analyses were performed for the target genes. Comparison of libraries showed that Wolbachia infection significantly regulated 91 miRNAs in females and 20 miRNAs in males, with an overall suppression of miRNAs in Wolbachia-infected libraries. A comparison of the miRNA and mRNA data predicted that the differentially expressed miRNAs negatively regulated 90 mRNAs in females and 9 mRNAs in males. An analysis of target genes showed that Wolbachia-responsive miRNAs regulated genes with function in sphingolipid metabolism, lysosome function, apoptosis and lipid transporting in both sexes, as well as reproduction in females.

Conclusion

Comparisons of the miRNA and mRNA data can help to identify miRNAs and miRNA target genes involving in Wolbachia-host interactions. The molecular targets identified in this study should be useful in further functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1122) contains supplementary material, which is available to authorized users.