Ectopically expressed Slc34a2a sense-antisense transcripts cause a cerebellar phenotype in zebrafish embryos depending on RNA complementarity and Dicer

Abstract

Natural antisense transcripts (NATs) are complementary to protein coding genes and potentially regulate their expression. Despite widespread occurrence of NATs in the genomes of higher eukaryotes, their biological role and mechanism of action is poorly understood. Zebrafish embryos offer a unique model system to study sense-antisense transcript interplay at whole organism level. Here, we investigate putative antisense transcript-mediated mechanisms by ectopically co-expressing the complementary transcripts during early zebrafish development. In zebrafish the gene Slc34a2a (Na-phosphate transporter) is bi-directionally transcribed, the NAT predominantly during early development up to 48 hours after fertilization. Declining levels of the NAT, Slc34a2a(as), coincide with an increase of the sense transcript. At that time, sense and antisense transcripts co-localize in the endoderm at near equal amounts. Ectopic expression of the sense transcript during embryogenesis leads to specific failure to develop a cerebellum. The defect is RNA-mediated and dependent on sense-antisense complementarity. Overexpression of a Slc34a2a paralogue (Slc34a2b) or the NAT itself had no phenotypic consequences. Knockdown of Dicer rescued the brain defect suggesting that RNA interference is required to mediate the phenotype. Our results corroborate previous reports of Slc34a2a-related endo-siRNAs in two days old zebrafish embryos and emphasize the importance of coordinated expression of sense-antisense transcripts. Our findings suggest that RNAi is involved in gene regulation by certain natural antisense RNAs.

A cautionary tale of sense-antisense gene pairs: independent regulation despite inverse correlation of expression

“…At the level of transcription, they can induce promoter methylation ( 16 – 18 ), recruit histone modifying enzymes ( 19 – 25 ), directly interfere and block the transcriptional machinery via transcriptional interference ( 26 – 29 ) or regulate sense mRNA splicing ( 30 – 32 ). Post-transcriptionally, they can bind to their sense mRNA and increase its stability by masking miRNA binding sites ( 33 ) or enhance its translation by recruiting additional factors ( 34 ) or generate endogenous siRNAs from double-stranded sense-antisense-hybrids ( 35 , 36 ). Genome-wide expression analysis of sense-antisense pairs has indicated that these are generally positively correlated ( 37 ).…”

Monoallelic, antisense and total RNA transcription in an in vitro neural differentiation system based on F1 hybrid mice

“…Functional generalization of antisense transcripts remains elusive, although intense investigation has been carried out since the report of pervasive antisense transcription in the mouse genome and likeliness of their functionality (Kiyosawa et al, 2003(Kiyosawa et al, , 2005. Anti-correlation of expression between sense and antisense transcripts has been observed in individual gene loci, in some of which disruption of the expression of the antisense or sense transcript affected specific phenotypes (Janowski et al, 2007;Morris et al, 2008;Yu et al, 2008;Watts et al, 2010;Modarresi et al, 2012;Piatek et al, 2017). However, a high-throughput experiment revealed mostly positive correlation between sense and antisense transcript expression (Cawley et al, 2004).…”

“…However, a high-throughput experiment revealed mostly positive correlation between sense and antisense transcript expression (Cawley et al, 2004). In addition, previous studies have reached a consensus that antisense transcripts form several heterogeneous groups with different functions (Faghihi and Wahlestedt, 2009;Piatek et al, 2017). The clear demarcation of developmental-stage-specific and constitutive fractions of antisense transcripts, the biases of positively and negatively correlated variation in expression of sense and antisense transcripts between different combinations of poly(A)+ and total RNA transcripts, and the location and size of the transcript overlap, support this consensus.…”

“…At present no categorization can segregate the sense and antisense transcript pairs of negative and positive expression variation completely. Additional structural (of the 5′ and 3′ end) (Werner et al, 2009) and functional analyses in conjunction with the proposed mechanisms involving both the 5′ and 3′ end of sense transcripts (summarized in Piatek et al, 2017) is expected to improve the demarcation of the positive and negative regulatory relationship.…”

Natural Antisense Transcripts at the Interface between Host Genome and Mobile Genetic Elements

“…The strategies to adopt a similar approach in mammalian cells, however, failed almost completely. Only a few cell types, oocytes or certain embryonic cells, seem to tolerate significant levels of dsRNA without triggering an immune response ( Wianny and Zernicka-Goetz, 2000 ; Piatek et al, 2017 ). As a consequence the contribution of Dicer and RNA interference to the processing of natural sense/antisense transcript pairs is controversial.…”