## 2 Results  
### Upregulation of Inc-Nr6a1 and Embedded miR-181a2 and miR-181b2 during EMT  
To systematically identify lncRNAs involved early in the EMT process, transcriptional profiling was performed in untreated NMuMG mammary epithelial cells and NMuMG cells treated with TGF-\(\beta\) for 2 h using microarrays that covered 31,423 annotated lncRNA-NAs and also 25,376 protein-coding transcripts [5]. Among PCR-confirmed candidates, _Nr6a1 os_ was one of the most significantly induced non-coding RNAs. This gene maps on chromosome 2 and is transcribed from the third intron of the _Nr6a1_ gene in the opposite sense. Two isoforms of 650 bp and 645 bp, here named Inc-_Nr6a1-1_ and Inc-_Nr6a1-2_, respectively, were induced early after TGF-\(\beta\) treatment. The isoform Inc-_Nr6a1-1_ is composed of three exons separated by introns of 16,600 and 13,806 bp, and Inc-_Nr6a1-2_ of two exons and an intron of 14,699 bp. MiR-181a2 and miR-181b2 genes are located at 314 and 1410 bp downstream, respectively, of the 3\({}^{\prime}\) end of Inc-_Nr6a1-1_ (Figure 1A). Exon2- and miR-181a2/b2-specific probes showed that an unprocessed Inc-pri-miR-181a2/b2 transcript was generated containing spliced exon1, exon2 and exon3 and an unprocessed fragment with embedded miR-181a2 and miR-181b2 genes (Figure 1A-C). Quantitative reverse-transcription PCR (RT-qPCR) of poly(A\({}^{\star}\))- and poly(A\({}^{-}\))-selected RNA showed that the Inc-_Nr6a1-1_ isoform was found in both RNA populations, in contrast to the Inc-_Nr6a1-2_ isoform that was found only in the poly(A\({}^{\star}\)) RNA population. We validated the enrichment using primers specific for a polyadenylated mRNA (_Hprt_) and non-polyadenylated RNA (_U6_) (Figure 1D). To map Inc-pri-miR-181a2/b2 3\({}^{\prime}\) ends at nucleotide resolution, we developed both a poly(A\({}^{\star}\)) tail-dependent and a poly(A\({}^{\star}\)) tail-independent 3\({}^{\prime}\) rapid amplification of cDNA ends (3\({}^{\prime}\) RACE) technique. Two poly(A\({}^{\star}\))-dependent polyadenylation sites were mapped, one of them located after the second exon of the Inc-_Nr6a1-2_ isoform, according to the annotation ENSMUST0000129089.2 from GENECODE, with a canonical AATAA polyadenylation signal occurring 35 nt upstream from the poly(A) tail, and the other one located after the third exon of Inc-_Nr6a1-1_, 49 nucleotides longer than the ENSMUST00000151229.2 transcript annotation, with a noncanonical AATAAAA polyadenylation signal occurring 19 nt upstream from the poly(A) tail (Figure S1A). Four poly(A\({}^{\star}\)) tail-independent polyadenylation sites were mapped, corresponding to the sites of Drosha cleavage on the 5\({}^{\prime}\) arm of the pre-miR-181a2 and pre-miR-181b2, respectively; and the site of Drosha cleavage on the 3\({}^{\prime}\) arm of the pre-miR-181a2 and pre-miR-181b2, respectively (Figures S1A and S2A,B). These results indicate that the Inc-_Nr6a1-1_ isoform is transcribed as a polyadenylated RNA and also as a precursor non-polyadenylated RNA, which would be processed by the Microprocessor complex providing pre-miR-181a2 and pre-miR-181b2 molecules and a IncRNA molecule that would be degraded since it is not detected using specific primers. We determined by 5\({}^{\prime}\) rapid amplification of cDNA ends (5\({}^{\prime}\) RACE) that Inc-_Nr6a1-1_ is a transcript 75 nucleotides longer at the 5\({}^{\prime}\) end than the ENSMUST00000151229.2 transcript annotation (Figures S1B and 2C,D). _Luc-Nr6a1_ is located at the third intron of the _Nr6a1_ gene, and its transcription could play a _cis_-regulatory role in the expression of this gene. However, _Nr6a1_ expression, unlike Inc-_Nr6a1_ isoforms and miR-181a2/b2, is hardly affected by treatment with TGF-\(\beta\) (Figure 1E-G).