Figure 7a shows the predicted complementary sequences of U3snoRNA and AKAP9 3 untranslated region (UTR)

Figure 7a shows the predicted complementary sequences of U3snoRNA and AKAP9 3 untranslated region (UTR). Open in a separate window Figure 7 Expression regulation of a reporter construct containing the 3 untranslated region (UTR) of the A-kinase anchor protein 9 (AKAP9) K145 mRNA by U3snoRNA and binding of U3snoRNA to Argonaute 2 (Ago2); (a) Predicted complementary sequences of U3snoRNA and AKAP9 3UTR; (b) Control of the expression of enhanced green fluorescent protein (EGFP) by U3snoRNA via the 3UTR of AKAP9 mRNA linked to the K145 EGFP-encoding cDNA (pCIneo-EGFP-3-UTR-AKAP9). U3snoRNA interacts with Argonaute 2 in the RNA-induced silencing complexes (RISC), pointing to a microRNA-like function. For this reason, the 3 untranslated region of the A-kinase anchor protein 9 (AKAP9)-mRNA could be identified as a potential target. [1]. Some others are involved in the mediation of metabolic stress [9], the regulation of alternative splicing [10], or the negative regulation of gene expression [11,12]. In contrast to most snoRNA genes found within introns of host genes [13,14], those of U3-, U8-, and U13snoRNAs are self-developed and have their own promoters for independent transcription [15]. The knowledge about their expression regulation, however, is sparse. The DEAD box proteins Ddx5 (p68) and Ddx17 (isoforms p72 and p82) modulate RNA secondary structures and are involved in RNA metabolic processes like ribosome biosynthesis and splicing [16,17,18,19]. They also act as transcriptional cofactors, repressing some promoters and activating others by interaction, e.g., with the histone deacetylase 1 (HDAC1) [20] or estrogen receptor alpha, respectively [21]. Investigating the role of Ddx5/Ddx17 in the processing of 28S rRNA, we observed that these proteins also influence the cellular level of U8snoRNA in HeLa cells [19]. Here, the scope of these investigations was expanded to the expression of further snoRNAs, notably those encoded by autonomous genes. We found a distinct inhibition of U3-, U8-, and U13snoRNA transcription by Ddx5/Ddx17. The inhibition is most probably mediated by the interactions with promoter bound HDAC1. In addition, HeLa cell proliferation and viability are shown to be dependent on the presence of U3snoRNA, and thus Ddx5/Ddx17 may negatively regulate these processes by their expression control. Furthermore, we show for the first time that U3snoRNA has a microRNA (miRNA)-like function, and the A-kinase anchor protein 9 (AKAP 9)-mRNA was identified as one of its potential targets. 2. Results 2.1. Ddx5 Regulates the Expression of U3-, U8-, and U13snoRNA We have shown previously that the cellular concentration of U8snoRNA increases significantly with reduced levels of Ddx5 and Ddx17 in HeLa cells [19]. Here we have investigated whether this observation can be extended to the expression of other snoRNAs encoded by autonomous genes such as U3- and U13snoRNA, that are involved in the processing of the 18S pre-rRNA [22]. Because Ddx5 and Ddx17 are highly related and are both active as transcriptional regulators [20], we used a small interfering RNA (siRNA) targeting both DEAD box proteins in knockdown experiments (Figure 1c). We observed an up-regulation of the cellular content of the U3-, U8-, and U13snoRNA upon Ddx5/17 siRNA transfection increasing with HD3 time and reaching a plateau after 40C48 h (Figure 1a). In contrast, the expression of the U14snoRNA, which is also involved in the processing of pre-rRNA but encoded in the intron of the ribosomal protein S13 (Rps13) and hence not transcribed independently [1], was not affected. Off-target effects of K145 the Ddx5/17si can be excluded by use of different siRNAs in the analysis of U8snoRNA expression in previous studies [19]. Overexpression of Ddx5 from an exogenous plasmid strongly repressed U3-, U8-, and U13snoRNA expression, whereas that of U14snoRNA was not affected (Figure 1b,d). Open in a separate window Figure 1 Effect of DEAD box proteins Ddx5/Ddx17 on the expression of U3-, U8-, U13-, and U14 small nucleolar RNA (snoRNA). (a) Northern blot analysis after knockdown of Ddx5/Ddx17. Total RNA (5 g each) from HeLa cells transfected with indicated small interfering RNA (siRNA) for 48 h were analyzed using digoxigenin-labeled snoRNA probes and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA probe, the latter serving as an endogenous loading control; (b) Northern blot analysis after overexpression of Ddx5. HeLa cells were transfected with a Ddx5 expression plasmid (Ddx5-tag) for 48 h, thereafter the total RNA was extracted and analyzed as in Figure 1a; (c) Demonstration of efficient Ddx5/Ddx17 knockdown by Western blot analysis with -tubulin as an endogenous loading control; (d) Demonstration of ectopic Ddx5 expression (Ddx5-myc) by Western blot analysis with myc antibodies. -Tubulin was used as an endogenous.