Target genes can be silenced by transfection of chemically or enzymatically synthesized small interfering RNAs (siRNA) or by DNA-based vector systems that encode short hairpin RNAs (shRNAs) that are further processed into siRNAs in the cytoplasm. The initially designed and most widely used vector-based RNA interferences (RNAi) are driven by RNA polymerase III promoters, e.g., H1 and U6 [1,2]. Several recent RNAi vectors driven by polymerase II promoters are based on endogenous small RNAs ( 22 nucleotides) known as microRNAs (miRNAs) that can also guide cleavage of RNAs and ⁄ or translational inhibition. Cullen and colleagues first described this kind of RNAi vector in which a synthetic siRNA ⁄ miRNA is expressed from a synthetic stem-loop precursor based on the miR30 miRNA precursor [3]. Subsequently, other groups have developed additional miR30-or miR155-based vectors for RNAi [4][5][6]. The expression of siRNAs from the artificial miRNA driven by an RNA polymerase II promoter offers several advantages over an RNA polymerase III promoter, including expression of several artificial miRNAs from a single transcript, and tissue-specific or regulated expression [4,6,7].In animals, primary miRNAs (pri-miRNAs) are transcribed by RNA polymerase II, and contain 5¢ CAP structures and 3¢ poly(A) tails [8,9]. The primiRNA is recognized and cleaved at a specific hairpin site by the nuclear microprocessor complex, which contains an RNase III family enzyme, Drosha, to produce a miRNA precursor (pre-miRNA) of approximately 70-90 nucleotides with a 2 nucleotide 3¢ overhang [10][11][12][13][14]. This distinctive structure activates transport of the pre-miRNA to the cytoplasm by 9,15]. RNA interference (RNAi) mediates sequence-specific post-transcriptional gene silencing in many eukaryotes and is used for reverse genetic studies and therapeutics. RNAi is triggered by double-stranded small interfering RNAs (siRNAs), which can be processed from small hairpin RNAs generated from an expression vector. In some recently described vectors, the siRNAs are expressed from synthetic stem-loop precursors of microRNAs (miRNAs) driven by polymerase II promoters. We have designed new RNAi vectors, designated pSM155 and pSM30, that take into consideration miRNA processing and RNA splicing by placing the miRNA-based artificial miRNA expression cassettes inside of synthetic introns. Like the original miRNA vectors, we show that the pSM155 and pSM30 constructs efficiently down-regulate expression of firefly luciferase and an endogenous gene, phospholipase D2. Moreover, the expression of a coexpressed fluorescent marker is substantially improved by this new design. Another improvement of these new vectors is incorporation of two inverted BsmBI sites placed internal to the arms of the new miRNA-based vectors, so oligos used for cloning are shorter and the cost is reduced. These RNAi vectors thus provide new tools for gene suppression.Abbreviations EGFP, enhanced green fluorescent protein; miRNA, microRNA; pre-miRNA, miRNA precursor; PLD2, phospholipa...

We previously reported that phospholipase C-␦ 1 (PLC-␦ 1 ) accumulates in the nucleus at the G 1 /S transition, which is largely dependent on its binding to phosphatidylinositol 4,5-bisphosphate (Stallings, J. D., Tall, E. G., Pentyala, S., and Rebecchi, M. J. (2005) J. Biol. Chem. 280, 22060 -22069). Here, using small interfering RNA (siRNA) that specifically targets rat PLC-␦1, we investigated whether this enzyme plays a role in cell cycle control. Inhibiting expression of PLC-␦ 1 significantly decreased proliferation of rat C6 glioma cells and altered S phase progression. [3 H]Thymidine labeling and fluorescence-activated cell sorting analysis indicated that the rates of G 1 /S transition and DNA synthesis were enhanced. On the other hand, knockdown cultures released from the G 1 /S boundary were slower to reach full G 2 /M DNA content, consistent with a delay in S phase. The levels of cyclin E, a key regulator of the G 1 /S transition and DNA synthesis, were elevated in asynchronous cultures as well as those blocked at the G 1 /S boundary. Epifluorescence imaging showed that transient expression of human phospholipase C-␦ 1 , resistant to these siRNA, suppressed expression of cyclin E at the G 1 /S boundary despite treatment of cultures with rat-specific siRNA. Although whole cell levels of phosphatidylinositol 4,5-bisphosphate were unchanged, suppression of PLC-␦ 1 led to a significant rise in the nuclear levels of this phospholipid at the G 1 /S boundary. These results support a role for PLC-␦ 1 and nuclear phospholipid metabolism in regulating cell cycle progression.Phosphoinositides are metabolized by a multifaceted and highly regulated set of phosphoinositide-specific enzymes (2-4). Kinases sequentially phosphorylate the inositol headgroup of phosphatidylinositol, and phosphatases reverse this process (5-7); both can generate phosphatidylinositol 4,5-bisphosphate (PIP 2 ), 2 the principle substrate of phospholipase C (PLC) (2-4). PLC cleaves PIP 2 to generate key second messengers, inositol 1,4,5-trisphosphate and diacylglycerol (2-4), that mobilize internal calcium stores (8) and activate protein kinase C (9), respectively. In mammals, the PLC family consists of at least 13 isoforms that fall into six subtypes: ␤, ␥, ␦, ⑀ (2-4), (10), and (11, 12). PIP 2 hydrolysis is vital to a wide range of cellular responses, including cytoskeletal remodeling (13), membrane trafficking (14), and gene transcription (15), proliferation, and differentiation (3, 4). PIP 2 metabolism and PLC activity (particularly in the nucleus) play prominent roles in cell cycle progression and ultimately influence global decisions, such as differentiation and proliferation (6, 16 -18). Indeed, homozygous deletion of PLC␤ 3 (19) or PLC␥ 1 (20) is embryonic lethal. Although PLC␦ 1 is not essential, homozygous deletion in mice results in aberrant expression of terminal differentiation markers in several types of skin cells as well as the development of alopecia and spontaneous skin tumors (21). These effects appear to result from increas...

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