Exploring the functions of RNA interference pathway proteins: some functions are more RISCy than others?

PPD (PAZ Piwi domain) proteins and the Dicer family have been the subjects of intense study over the last 6 years. These proteins have well-established roles in RNAi (RNA interference), a process that relies on siRNAs (small interfering RNAs) or miRNAs (microRNAs) to mediate specificity. The development of techniques for applying RNAi as a laboratory tool and a molecular therapeutic technique has rapidly outpaced our understanding of the biology of this process. However, over the last 2 years, great strides have been made towards elucidating how PPD proteins and Dicer regulate gene-silencing at the pre- and post-transcriptional levels. In addition, evidence is beginning to emerge that suggests that these proteins have additional siRNA-independent roles as cell-cycle regulators. In the present review, we summarize the well-known roles of these two classes of proteins in gene-silencing pathways, as well as explore the evidence for novel roles of PPD and Dicer proteins.

Adenovirus-5 E1A suppresses differentiation of 3T3 L1 preadipocytes at lower levels than required for induction of apoptosis.

To investigate the functional relationship between the ability of the adenovirus-5 E1A oncogene product to transform with its ability to block adipocytic differentiation and induce apoptosis, we expressed E1A in the 3T3 L1 preadipocytic cell line. The results demonstrate a dramatic, quantitative reciprocal regulation of differentiation and several transformation-associated properties in response to graded levels of E1A expression, with the suppression of differentiative capacity, focus formation, and anchorage-independent proliferation requiring increasing levels of E1A. Progressively higher E1A levels were accompanied by apoptosis induction. The effect of E1A upon adipocytic differentiation as well as transformation and apoptosis required binding to the retinoblastoma-susceptibility gene product. These data reveal a dissociation between E1A signals leading to transformation, suppression of differentiation and induction of apoptosis, based on levels of expression.

Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer.

PAZ PIWI domain (PPD) proteins, together with the RNA cleavage products of Dicer, form ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs). RISCs mediate gene silencing through targeted messenger RNA cleavage and translational suppression. The PAZ domains of PPD and Dicer proteins were originally thought to mediate binding between PPD proteins and Dicer, although no evidence exists to support this theory. Here we show that PAZ domains are not required for PPD protein-Dicer interactions. Rather, a subregion of the PIWI domain in PPD proteins, the PIWI-box, binds directly to the Dicer RNase III domain. Stable binding between PPD proteins and Dicer was dependent on the activity of Hsp90. Unexpectedly, binding of PPD proteins to Dicer inhibits the RNase activity of this enzyme in vitro. Lastly, we show that PPD proteins and Dicer are present in soluble and membrane-associated fractions, indicating that interactions between these two types of proteins may occur in multiple compartments.