While my yard is recovering from the ice, and I from today’s UK game, I thought I would toss out a few interesting abstracts that touch on important and contentious issues. Peek beneath the fold and, as always, enjoy.
From an article by Fox-Walsh and Hertel, a study that suggests that so-called alternative splicing is going to occur in most genes with multiple introns, but that splicing is inherently accurate, meaning that alternative splicing is likely to be quite amenable (or accessible) to control mechanisms.
“The extensive alternative splicing in higher eukaryotes has initiated a debate whether alternative mRNA isoforms are generated by an inaccurate spliceosome or are the consequence of highly degenerate splice sites within the human genome. Here, we established a quantitative assay to evaluate the accuracy of splice-site pairing by determining the number of incorrect exon-skipping events made from constitutively spliced pre-mRNA transcripts. We demonstrate that the spliceosome pairs exons with an astonishingly high degree of accuracy that may be limited by the quality of pre-mRNAs generated by RNA pol II. The error rate of exon pairing is increased by the effects of the neurodegenerative disorder spinal muscular atrophy because of reduced levels of Survival of Motor Neuron, a master assembler of spliceosomal components. We conclude that all multi-intron-containing genes are alternatively spliced and that the reduction of SMN results in a general splicing defect that is mediated through alterations in the fidelity of splice-site pairing.”
As I discussed before, alternative polyadenylation is an important regulatory process. However, with precious few exceptions, it is not clear how signaling input is manifest as a change in polyadenylation. A study from Kevin Ryan’s lab has implications for the control/regulation of polyadenylation, as it implicates phosphorylation/dephosphorylation of protein subunits in the modulation of activity of the polyadenylation complex. The abstract:
“3′ Cleavage and polyadenylation are obligatory steps in the biogenesis of most mammalian pre-mRNAs. In vitro reconstitution of the 3′ cleavage reaction from human cleavage factors requires high concentrations of creatine phosphate (CP), though how CP activates cleavage is not known. Previously, we proposed that CP might work by competitively inhibiting a cleavage-suppressing serine/threonine (S/T) phosphatase. Here we show that fluoride/EDTA, a general S/T phosphatase inhibitor, activates in vitro cleavage in place of CP. Subsequent testing of inhibitors specific for different S/T phosphatases showed that inhibitors of the PPM family of S/T phosphatases, which includes PP2C, but not the PPP family, which includes PP1, PP2A, and PP2B, activated 3′ cleavage in vitro. In particular, NCI 83633, an inhibitor of PP2C, activated extensive 3′ cleavage at a concentration 50-fold below that required by fluoride or CP. The testing of structural analogs led to the identification of a more potent compound that activated 3′ cleavage at 200 μM. While testing CP analogs to understand the origin of its cleavage activation effect, we found phosphocholine to be a more effective activator than CP. The minimal structural determinants of 3′ cleavage activation by phosphocholine were identified. Our results describe a much improved small molecule activator of in vitro pre-mRNA cleavage, identify the molecular determinants of cleavage activation by phosphoamines such as phosphocholine, and suggest that a PPM family phosphatase is involved in the negative regulation of mammalian pre-mRNA 3′ cleavage.”
As discussed here, target recognition by microRNAs involves a relatively small “seed” region; this has significant ramifications as far as the extent to which therapeutic microRNAs might affect non-target transcripts. A recent report by Burchard et al. suggests that siRNAs are affected by similar considerations.
“siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand “seed region,” similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models.”
Finally, a report by Lebreton et al. that reveals that the RNA-degrading exosome complex has endonuclease activity as well as the well-known exonuclease activities. This report brings to my mind a fascinating (and possibly meaningful) parallel with the metallo-beta-lactamase family of nucleases (such as CPSF73 or RNAse J).
“The exosome is a major eukaryotic nuclease located in both the nucleus and the cytoplasm that contributes to the processing, quality control and/or turnover of a large number of cellular RNAs. This large macromolecular assembly has been described as a 3′→5′ exonuclease and shown to contain a nine-subunit ring structure evolutionarily related to archaeal exosome-like complexes and bacterial polynucleotide phosphorylases. Recent results have shown that, unlike its prokaryotic counterparts, the yeast and human ring structures are catalytically inactive. In contrast, the exonucleolytic activity of the yeast exosome core was shown to be mediated by the RNB domain of the eukaryote-specific Dis3 subunit. Here we show, using in vitro assays, that yeast Dis3 has an additional endoribonuclease activity mediated by the PIN domain located at the amino terminus of this multidomain protein. Simultaneous inactivation of the endonucleolytic and exonucleolytic activities of the exosome core generates a synthetic growth phenotype in vivo, supporting a physiological function for the PIN domain. This activity is responsible for the cleavage of some natural exosome substrates, independently of exonucleolytic degradation. In contrast with current models, our results show that eukaryotic exosome cores have both endonucleolytic and exonucleolytic activities, mediated by two distinct domains of the Dis3 subunit. The mode of action of eukaryotic exosome cores in RNA processing and degradation should be reconsidered, taking into account the cooperation between its multiple ribonucleolytic activities.”
Fox-Walsh KL, Hertel KJ. Splice-site pairing is an intrinsically high fidelity process. Proc Natl Acad Sci U S A. 2009 Jan 29. [Epub ahead of print]
Ryan K, Khleborodova A, Pan J, Ryan XP. Small molecule activators of pre-mRNA 3′ cleavage. RNA. 2009 Jan 20. [Epub ahead of print]
Burchard J, Jackson AL, Malkov V, Needham RH, Tan Y, Bartz SR, Dai H, Sachs AB, Linsley PS. MicroRNA-like off-target transcript regulation by siRNAs is species specific. RNA. 2009 Feb;15(2):308-15. doi: 10.1261/rna.1326809
Lebreton A, Tomecki R, Dziembowski A, Séraphin B. Endonucleolytic RNA cleavage by a eukaryotic exosome. Nature. 2008 Dec 18;456(7224):993-6. Epub 2008 Dec 7. doi:10.1038/nature07480