Mechanisms of RNAi: mRNA cleavage fragments may indicate stalled RISC

The molecular mechanism of RNA interference (RNAi) is under intense investigation. We previously demonstrated the existence of inactive siRNAs and also of mRNA cleavage in vivo in human cells. Here it is shown that some siRNAs with low activity leave mRNA cleavage fragments while an siRNA with higher activity does not. The pattern is consistent with both short-term (4-24 hours) and long-term (1-4 days) time-series. Analysis of the putative 3′ mRNA cleavage product showed high GC content immediately after the cleavage point. The cleavage fragments might indicate a stalled or slowed RNAi cleavage complex - possibly in the RISC enzyme restoration phase - and thus constitute a novel explanation for the existence of inactive siRNAs

A cryptic targeting signal creates a mitochondrial FEN1 isoform with tailed R-Loop binding properties.

A growing number of DNA transacting proteins is found in the nucleus and in mitochondria, including the DNA repair and replication protein Flap endonuclease 1, FEN1. Here we show a truncated FEN1 isoform is generated by alternative translation initiation, exposing a mitochondrial targeting signal. The shortened form of FEN1, which we term FENMIT, localizes to mitochondria, based on import into isolated organelles, immunocytochemistry and subcellular fractionation. In vitro FENMIT binds to flap structures containing a 5' RNA flap, and prefers such substrates to single-stranded RNA. FENMIT can also bind to R-loops, and to a lesser extent to D-loops. Exposing human cells to ethidium bromide results in the generation of RNA/DNA hybrids near the origin of mitochondrial DNA replication. FENMIT is recruited to the DNA under these conditions, and is released by RNase treatment. Moreover, high levels of recombinant FENMIT expression inhibit mtDNA replication, following ethidium bromide treatment. These findings suggest FENMIT interacts with RNA/DNA hybrids in mitochondrial DNA, such as those found at the origin of replication

Comparative regulomics supports pervasive selection on gene dosage following whole genome duplication

publishedVersio

Efficient prediction of siRNAs with siRNArules 1.0: An open-source JAVA approach to siRNA algorithms

RNAi interference and siRNA have become useful tools for investigation of gene function. However, the discovery that not all siRNA are equally efficient made necessary screens or design algorithms to obtain high activity siRNA candidates. Several algorithms have been published, but they remain inefficient, obscure, or commercially restricted. This article describes an open-source JAVA program that is surprisingly efficient at predicting active siRNAs (Pearson correlation coefficient r = 0.52, n = 526 siRNAs). Furthermore, this version 1.0 sets the stage for further improvement of the free code by the open-source community (http://sourceforge.net/)

New isoforms of rat Aquaporin-4.

AbstractAquaporin-4 (AQP4) is a brain aquaporin implicated in the pathophysiology of numerous clinical conditions including brain edema. Here we show that rat AQP4 has six cDNA isoforms, formed by alternative splicing. These are named AQP4a–f, where AQP4a and AQP4c correspond to the two classical M1 and M23 isoforms, respectively. The various isoforms are differentially expressed in kidney and brain, and their prevalence does not correspond to the level of the respective mRNAs, pointing to posttranscriptional regulation. The three isoforms lacking exon 2, AQP4b, AQP4d, and AQP4f, have an intracellular localization when expressed in cell lines and do not transport water when expressed in Xenopus oocytes. In contrast, the largest of the new isoforms, AQP4e, which contains a novel N-terminal domain, is localized at the plasma membrane in cell lines and functions as a water transporter in Xenopus oocytes

Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo

in viv