Exploring the function of long non-coding RNA in the development of bovine early embryosby Julieta Caballero, Isabelle Gilbert, Eric Fournier, Dominic Gagn�, Sara Scantland, Angus Macaulay, Claude Robert

Reprod. Fertil. Dev.



Exploring the function of long non-coding RNA in the development of bovine early embryos

Julieta CaballeroA, Isabelle GilbertA, Eric FournierA, Dominic Gagne´A,

Sara ScantlandA, Angus MacaulayA and Claude RobertA,B

ADe´partement des sciences animales, Centre de recherche en biologie de la reproduction,

Institut sur la nutrition et les aliments fonctionnels, Universite´ Laval, 2440 boulevard Hochelaga QC, G1V 0A6, Canada.

BCorresponding author. Email: claude.robert@fsaa.ulaval.ca

Abstract. Now recognised as part of the cellular transcriptome, the function of long non-coding (lnc) RNA remains unclear. Previously, we found that some lncRNAmolecules in bovine embryos are highly responsive to culture conditions.

In view of a recent demonstration that lncRNA may play a role in regulating important functions, such as maintenance of pluripotency, modification of epigenetic marks and activation of transcription, we sought evidence of its involvement in embryogenesis. Among the numerous catalogued lncRNA molecules found in oocytes and early embryos of cattle, three candidates chosen for further characterisation were found unexpectedly in the cytoplasmic compartment rather than in the nucleus. Transcriptomic survey of subcellular fractions found these candidates also associated with polyribosomes and one of them spanning transzonal projections between cumulus cells and the oocyte. Knocking down this transcript in matured oocytes increased developmental rates, leading to larger blastocysts. Transcriptome and methylome analyses of these blastocysts showed concordant data for a subset of four genes, including at least one known to be important for blastocyst survival. Functional characterisation of the roles played by lncRNA in supporting early development remains elusive. Our results suggest that some lncRNAs play a role in translation control of target mRNA. This would be important for managing the maternal reserves within which is embedded the embryonic program, especially before embryonic genome activation.

Additional keywords: cytoplasm, knockdown, methylome, polyribosome, transcriptome, transzonal projection.


Thanks to advances in high-throughput RNA sequencing, it has been found that a very large portion of the human genome is transcribed, much more than the 1%–3% that encodes proteins (Djebali et al. 2012; Hangauer et al. 2013). In fact, approximately 83.5% is transcribed (Djebali et al. 2012), with the residual 80% consisting of non-coding RNA, sequences corresponding to introns, cis-regulatory elements and repetitive elements (Cordaux and Batzer 2009). This has revealed a far more complex transcriptome than what was originally expected. Until recently, non-coding RNA had been regarded as genomic noise produced by a ‘leaky’ transcriptional apparatus, with no functional purpose and hence often called the genomic ‘dark matter’. Although the role of non-coding RNA, such as ribosomal RNA (rRNA) and transfer RNA (tRNA), in translation has long been understood, it has become clear only in recent years that this category also includes versatile molecules that play essential roles in a variety of biological processes, such as modulation of gene expression and epigenetic status (for reviews, see Wilusz et al. 2009; Pauli et al. 2011; Morris 2012).

Long non-coding (lnc) RNA constitutes the largest class of non-coding RNA, with approximately 15 000 human lncRNA genes characterised so far (Derrien et al. 2012). It has been estimated that 17% of these transcripts remain within the nucleus, whereas approximately 4% are enriched in the cytoplasm (Derrien et al. 2012). A wide range of different types of lncRNA have been identified, most notably intergenic lncRNA (lincRNA), intronic lncRNA, which overlaps with an intronic region of a protein-coding gene, natural antisense transcripts (NATs), which originate from the DNA strand opposite the protein-coding strand, and pseudogenes, defined as ancestral copies of coding genes created by duplication or retrotransposition (Lapidot and Pilpel 2006; Huang et al. 2012; Rapicavoli et al. 2013). LncRNA shares some similarities with mRNA in terms of sequence length, transcription (usually dependent on

RNA polymerase II), splicing (98% are spliced) and polyadenylation (Guttman et al. 2009, 2010; Ba´nfai et al. 2012; Derrien


Reproduction, Fertility and Development, 2015, 27, 40–52 http://dx.doi.org/10.1071/RD14338

Journal compilation  IETS 2015 www.publish.csiro.au/journals/rfd et al. 2012). No protein has yet been traced back to them. They are generally poorly conserved between species. In humans, one-third of them appear to be primate specific, whereas 44% show some higher degree of evolutionary conservation (Derrien et al. 2012). Ranging in length from 200 to several thousand nucleotides, they play roles such as transcriptional regulators, molecular decoys, pairing with and guiding other RNA molecules, effectors, enhancers or activators, cytoplasmic scaffolds for transcription factors and epigenetic regulators (Mercer and

Mattick 2013; Ulitsky and Bartel 2013).

Current understanding of the roles of lncRNA in epigenetics has emerged from the study of Xist, the best characterised lncRNA, involved in X-chromosome inactivation in females (Brockdorff et al. 1991; Wutz and Gribnau 2007). In humans in particular, X-chromosome inactivation involves a set of lncRNA that includes Xist and its antisense transcript Tsix, as well as the more recently identified X-active transcript (Xact; Vallot et al. 2013). These molecules trigger a cascade of chromatin remodelling events that lead to DNA methylation and shut down transcription over most of the X-chromosome. Another prime example of gene expression control involving lncRNA is genomic imprinting. For several imprinted genes, memory of gender inheritance (which remains after fertilisation and drives either silencing or expression of one of the two parental alleles) also involves the expression of specific lncRNA molecules.