Testis-specific lncRNAs function in trans to regulate late spermatogenesis. (A) Male germ cell development defects of four lncRNA knockout mutants were rescued by restoration of the corresponding lncRNAs in cis or in trans. For in cis rescue, a construct bearing an lncRNA gene fragment under the control of the endogenous promoter was inserted back into the genomic locus from which the original lncRNA had been deleted. For rescue in trans, the lncRNA was placed on another chromosome (i.e., different from the original chromosomal locus of the lncRNA) through PhiC31-mediated attB/attP exchange. The abnormal morphological phenotype of scattered and curled sperm nuclei in late spermatogenesis of CR42858−/−, CR43484−/−, CR44585−/−, and lncRNA:TS2−/− were rescued by restoration of these lncRNAs either in cis or in trans. (B) LncRNA CR42858 was transcribed in flies rescued with CR42858 in cis and in trans, but not in CR42858−/−. RNA was isolated from the testes of wild-type, CR42858−/−, and flies rescued with CR42858 in cis or in trans, and subjected to semiquantitative RT-PCR. RpL32 was used as a control. (C) In cis or in trans rescue of CR42858 recovered the abnormal morphology of the testis, lower density of sperm, and lagging and poorly aligned IC phenotypes of CR42858−/−. (D) RNA-seq analysis of wild-type and CR42858−/− flies. The heat map shows fold changes in expression of each of the top 18 differentially expressed genes (DEGs) in CR42858−/− relative to those in the wild type, and the DEGs were corrected for the biological variability by combining biological replicates (NOISeqBIO method in NOISeq, version 2.14.1) (Tarazona et al. 2015). (E) Expression changes of six neighboring genes of CR42858−/−. RNA was isolated from testes of CR42858−/− and wild type and subjected to quantitative RT-PCR. RpL32 was used as an internal control. Results are represented as means ± SEM of three biological replicates. Asterisks indicate a significant difference between the samples (P < 0.05; t-test). (F) Schematic illustration of in vivo functions of lncRNAs in fly spermatogenesis. LncRNAs can regulate the expression of both local and distal genes in testis, and function in post-meiotic stages of spermatogenesis.
