Specifically, as shown in Figure 6B, CS 6 formed three hydrogen bonds with the

To date, a number of Dicer1 mRNA variants have been described. however, all the reported tran scripts have been found to encode the same full length protein. More recently, however, the first mRNA splice variant of the human dicer1 gene bearing a modified coding sequence was identified in AP24534 溶解度 neuroblast oma cells. In fact, the dicer1 gene has been pre dicted to produce 14 mRNA variants in addition to the one found in neuroblastoma cells, including 3 full length forms and 11 mRNA variants that encode truncated Dicer1 proteins of varying lengths, one being Dicer1e. Although our data, plus the Hinkal et al. study, had confirmed that a 93 kDa Dicer1 protein variant was present in cells, no biochemical evidence existed that this protein variant was the product of a predicted Dicer1 mRNA variant.

As a result, to confirm that cells expressed the Dicer1e transcript, we performed 5 and 3 RACE analyses using mRNA harvested from SCC 25 cells, a cell line exhibiting one of the high est levels of Dicer1e protein expression. 5 RACE analysis using a reverse primer designed to target a unique sequence found only in Dicer1e resulted in the amplification of a 380 bp AT7519 臨床試験 pro duct that closely corresponded to the expected 5 RACE product size of 373 bp. Subsequent 3 Dicer1e is predominantly localized in the nucleus Based on the NCBI AceView database, PSORT II analysis of the Dicer1e protein sequence predicts a possible bipartite NLS between amino acids 247 to 264 and the subcellular location of Dicer1e protein to be most likely in the nucleus. Furthermore, a more recent study by Doyle et al.

found that the dsRBD of human Dicer1 functions as a NLS, a domain that is also present in Dicer1e. Therefore, to investi gate the possibility that the Dicer1e protein was localized to the nucleus, we performed biochemical fractionation studies using cytoplasmic and nuclear protein extracts obtained from all the OSCC cell Alisertib Aurora キナーゼ 阻害剤 lines and HOKs. Ana lyses of the data demonstrated that Dicer1e was localized to RACE analysis using a forward primer, followed by nested PCR using a second forward primer designed to target the unique sequence of Dicer1e resulted in the amplification of a 2,500 bp product that also closely corresponded to the expected 3 RACE product size of 2,472 bp. Cloning and complete sequencing of both the 5 and 3 RACE products confirmed the existence of the Dicer1e tran script.

Of note, RT PCR analysis was also performed in an attempt to detect the Dicer1d mRNA variant. how ever, no predicted PCR products were observed in either oral cancer cells or the T47D breast cancer cell line. The absence of this transcript in cells cor roborated our Western blot results and most likely ex plained why the 113 kDa Dicer1d protein was not expressed in cells. The sequence of the complete Dicer1e cDNA and the predicted amino acid sequence are shown in Figure 3A. Comparison between the experimen tal and the NCBI AceView database predicted cDNA se quences, demonstrated only minor differences in the 5 and 3 UTR regions with no alterations to the Dicer1e protein coding sequence.