Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.

contributed equally to this work Duplexes of 21±23 nucleotide (nt) RNAs are the sequence-speci®c mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate ef®cient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3¢ overhangs were the most ef®cient triggers of sequence-speci®c mRNA degradation. Substitution of one or both siRNA strands by 2¢-deoxy or 2¢-O-methyl oligonucleotides abolished RNAi, although multiple 2¢-deoxynucleotide substitutions at the 3¢ end of siRNAs were tolerated. The target recognition process is highly sequence speci®c, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is de®ned by the 5¢ end of the guide siRNA rather than its 3¢ end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.

Summary The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. We performed a functional screen to identify specific miRNAs that function at synapses to control dendritic spine structure. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of Acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including G protein alpha subunits (Gα). RNAi-mediated knockdown of APT1 and expression of membrane-localized Gα both suppress spine enlargement caused by miR-138 inhibition, suggesting that APT1-regulated depalmitoylation of Gα might be an important downstream event of miR-138 function. Our results uncover a novel miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.

The RNA-Induced Silencing Complex (RISC) is a ribonucleoprotein particle composed of a single-stranded short interfering RNA (siRNA) and an endonucleolytically active Argonaute protein, capable of cleaving mRNAs complementary to the siRNA. The mechanism by which RISC cleaves a target RNA is well understood, however it remains enigmatic how RISC finds its target RNA. Here, we show, both in vitro and in vivo, that the accessibility of the target site correlates directly with the efficiency of cleavage, demonstrating that RISC is unable to unfold structured RNA. In the course of target recognition, RISC transiently contacts single-stranded RNA nonspecifically and promotes siRNA-target RNA annealing. Furthermore, the 5' part of the siRNA within RISC creates a thermodynamic threshold that determines the stable association of RISC and the target RNA. We therefore provide mechanistic insights by revealing features of RISC and target RNAs that are crucial to achieve efficiency and specificity in RNA interference.

CLP1 is the first discovered mammalian RNA kinase. However, its in vivo function has been entirely elusive. We have generated kinase-dead Clp1 (Clp1K/K) mice which exhibit a progressive loss of spinal motor neurons associated with axonal degeneration in peripheral nerves, denervation of neuromuscular junctions, and results in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of an entirely novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-tRNA. These tRNA fragments sensitize cells to oxidative stress-induced p53 activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1K/K mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.

microRNAs (miRNAs) are endogenous, noncoding 22-nucleotide RNA molecules that have recently emerged as fundamental, post-transcriptional regulators of cognate target gene expression. Many mammalian miRNAs are expressed in a tissue-specific manner, a phenomenon that has so far been attributed to transcriptional regulation. We here show by Northern blots and in situ hybridization experiments that the expression of mammalian miRNAs can be regulated at the post-transcriptional level. In particular, miR-138 is spatially restricted to distinct cell types, while its precursor, pre-miR-138-2, is ubiquitously expressed throughout all tissues analyzed. Furthermore, pre-miR-138-2 is exported from the nucleus to the cytoplasm, suggesting that cleavage of this pre-miRNA by Dicer is restricted to certain tissues and cell types. Thus, differential processing of pre-miRNAs might be an alternative mechanism to control miRNA function.

The human enzyme that joins transfer RNA exons together is discovered.

Several microRNAs (miRNAs) have recently been described as crucial regulators of epithelial-to-mesenchymal transition (EMT) and metastasis. By comparing the expression profiles of miRNAs, we found upregulation of miR-29a in mesenchymal, metastatic RasXT cells relative to epithelial EpRas cells. Overexpression of miR-29a suppressed the expression of tristetraprolin (TTP), a protein involved in the degradation of messenger RNAs with AU-rich 3 0 -untranslated regions, and led to EMT and metastasis in cooperation with oncogenic Ras signalling. We also observed enhanced miR-29a and reduced TTP levels in breast cancer patient samples, indicating relevance for human disease. Previously, miR-29 family members were shown to have tumour-suppressive effects in haematopoietic, cholangiocytic and lung tumours. Therefore, miRNAs can act as either oncogenes or tumour suppressors, depending on the context.