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RNA interference (RNAi) is a eukaryotic gene-silencing system. Although the biochemistry of RNAi is relatively well defined, how this pathway is regulated remains incompletely understood. To identify genes involved in regulating the RNAi pathway, we screened for genetic mutations in Drosophila that alter the efficiency of RNAi. We identified the Drosophila homolog of the mammalian CR6-interacting factor 1 (CRIF1), also known as growth arrest and DNA-damage-inducible 45-gamma interacting protein (Gadd45GIP1), as a potential new regulator of the RNAi pathway. Loss-of-function mutants of Drosophila CRIF1 (dCRIF) are deficient in RNAi-mediated target gene knock-down, in the biogenesis of small interfering RNA (siRNA) molecules, and in antiviral immunity. Moreover, we show that dCRIF may function by interacting with, and stabilizing, the RNase III enzyme Dicer-2. Our results suggest that dCRIF may play an important role in regulating the RNAi pathway.

During normal translation, the binding of a release factor to one of the three stop codons (UGA, UAA or UAG) results in the termination of protein synthesis. However, modification of the initial uridine to a pseudouridine (Ψ) allows efficient recognition and read-through of these stop codons by a transfer RNA (tRNA), although it requires the formation of two normally forbidden purine-purine base pairs (1). Here we determined the crystal structure at 3.1 A resolution of the 30S ribosomal subunit in complex with the anticodon stem loop of [tRNA.sup.Ser] bound to the ΨAG stop codon in the A site. The Ψ A base pair at the first position is accompanied by the formation of purine-purine base pairs at the second and third positions of the codon, which show an unusual Watson-Crick/Hoogsteen geometry. The structure shows a previously unsuspected ability of the ribosomal decoding centre to accommodate non-canonical base pairs., The genetic code normally requires Watson-Crick base pairing at the first two positions of the codon-anticodon helix, and tolerates certain specific mismatches at the third (wobble) position. The structural basis [...]

Nonsense codons taken as read The three 'nonsense' or 'stop' codons that signal the ribosome to cease translation -- UAA, UAG and UGA -- all contain an initial uridine residue. John Karijolich and Yi-Tao Yu demonstrate that isomerization of this uridine, converting it to pseudouridine ([PSI]), allows the codons to be read by the ribosome in vivo. Specific amino acids are inserted into the resulting protein, with [PSI]AA and [PSI]AG coding for serine and threonine, and [PSI]GA for tyrosine and phenylalanine. Although there is yet no evidence for a physiological role for this RNA modification, it could offer a way of expanding the genetic code. Similar RNA modification could also be useful as a means of nonsense suppression in vivo, either experimentally or clinically. All three translation termination codons, or nonsense codons, contain a uridine residue at the first position of the codon.sup.1,2,3. Here, we demonstrate that pseudouridylation (conversion of uridine into pseudouridine ([PSI]), ref. 4) of nonsense codons suppresses translation termination both in vitro and in vivo. In vivo targeting of nonsense codons is accomplished by the expression of an H/ACA RNA capable of directing the isomerization of uridine to [PSI] within the nonsense codon. Thus, targeted pseudouridylation represents a novel approach for promoting nonsense suppression in vivo. Remarkably, we also show that pseudouridylated nonsense codons code for amino acids with similar properties. Specifically, [PSI]AA and [PSI]AG code for serine and threonine, whereas [PSI]GA codes for tyrosine and phenylalanine, thus suggesting a new mode of decoding. Our results also suggest that RNA modification, as a naturally occurring mechanism, may offer a new way to expand the genetic code., Author(s): John Karijolich [sup.1] , Yi-Tao Yu [sup.1] Author Affiliations: (1) Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, USA Main [PSI], the C5-glycoside isomer of uridine, [...]

Nematode trans-spliced leader (SL) RNAs are composed of two domains, an exon [the 22-nucleotide spliced leader] and a small nuclear RNA (snRNA)-like sequence. Participation in vitro of the spliced leader RNA in trans-splicing reactions is independent of the exon sequence or size and instead depends on features contained in the snRNA-like domain of the molecule. Chemical modification interference analysis has revealed that two short sequence elements in the snRNA-like domain are necessary for SL RNA activity. These elements are sufficient for such activity because when added to a 72-nucleotide fragment of a nematode U1 snRNA, this hybrid RNA could participate in trans-splicing reactions in vitro. One of the critical sequence elements may function by base-pairing with U6 snRNA, an essential U snRNA for both cis- and trans-splicing., In nematodes, a subset of mRNAs contains a common 5' terminal 22-nucleotide SL sequence. The SL sequence is acquired from a small ( 100 nucleotides) SL RNA by trans-splicing. The [...]