Scott Baskerville

3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets

Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3′ untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2–7 or 2–8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.

The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development

MicroRNAs (miRNAs) are an abundant class of gene regulatory molecules (reviewed in refs 1, 2). Although computational work indicates that miRNAs repress more than a third of human genes, their roles in vertebrate development are only now beginning to be determined. Here we show that miR-196 acts upstream of Hoxb8 and Sonic hedgehog (Shh) in vivo in the context of limb development, thereby identifying a previously observed but uncharacterized inhibitory activity that operates specifically in the hindlimb. Our data indicate that miR-196 functions in a fail-safe mechanism to assure the fidelity of expression domains that are primarily regulated at the transcriptional level, supporting the idea that many vertebrate miRNAs may function as a secondary level of gene regulation.

Anchoring an extended HTLV-1 Rex peptide within an RNA major groove containing junctional base triples

BACKGROUND The Rex protein of the human T cell leukemia virus type 1 (HTLV-1) belongs to a family of proteins that use arginine-rich motifs (ARMs) to recognize their RNA targets. Previously, an in vitro selected RNA aptamer sequence was identified that mediates mRNA transport in vivo when placed in the primary binding site on stem-loop IID of the Rex response element. We present the solution structure of the HTLV-1 arginine-rich Rex peptide bound to its RNA aptamer target determined by multidimensional heteronuclear NMR spectroscopy. RESULTS The Rex peptide in a predominantly extended conformation threads through a channel formed by the shallow and widened RNA major groove and a looped out guanine. The RNA aptamer contains three stems separated by a pair of two-base bulges, and adopts an unanticipated fold in which both junctional sites are anchored through base triple formation. Binding specificity is associated with intermolecular hydrogen bonding between guanidinium groups of three non-adjacent arginines and the guanine base edges of three adjacent G.C pairs. CONCLUSIONS The extended S-shaped conformation of the Rex peptide, together with previous demonstrations of a beta-hairpin conformation for the bovine immunodeficiency virus (BIV) Tat peptide and an alpha-helical conformation for the human immunodeficiency virus (HIV) Rev peptide in complex with their respective RNA targets, expands our understanding of the strategies employed by ARMs for adaptive recognition and highlights the importance of RNA tertiary structure in accommodating minimalist elements of protein secondary structure.

A ribozyme that ligates RNA to protein

We have used a combination of in vitro selection and rational design to generate ribozymes that form a stable phosphoamide bond between the 5′ terminus of an RNA and a specific polypeptide. This reaction differs from that of previously identified ribozymes, although the product is analogous to the enzyme–nucleotidyl intermediates isolated during the reactions of certain proteinaceous enzymes, such as guanyltransferase, DNA ligase, and RNA ligase. Comparative sequence analysis of the isolated ribozymes revealed that they share a compact secondary structure containing six stems arranged in a four-helix junction and branched pseudoknot. An optimized version of the ribozyme reacts with substrate-fusion proteins, allowing it to be used to attach RNA tags to proteins both in vitro and within bacterial cells, suggesting a simple way to tag a specific protein with amplifiable information.

Directed Evolutionary Descriptions of Natural RNA Structures

Selection experiments have frequently been used to optimize or alter the functions of biopolymers and have also proven useful for discerning biopolymer structure. Just as covariations observed in ribosomal RNA phylogeny may pinpoint Watson-Crick base pairs, sequence information recovered from in vitro and in vivo selection experiments can be used to establish and refine the structure of natural and unnatural nucleic acids. For example, RNA molecules selected from partially or completely randomized pools typically fall into a limited number of sequence families; the relationships within and between these families constitute an artificial phylogeny in which relationships are based on function rather than history. Nevertheless, comparative sequence analysis can still be used to identify what portions of primary sequence are essential for function, which residues potentially pair with one another, whether base pairs act as structural stabilizers or contribute directly to function, and even where tertiary structural interactions may occur. Such results can be combined to generate artificial evolutionary models for RNA structure. Although crystal or nuclear magnetic resonance (NMR) structures of molecules provide much greater spatial detail, evolutionary studies delve into function as well as structure and can provide insights into how structures may change through time. Evolutionary models can complement more physical determinations: For example, a detailed knowledge of the structure of rRNA or HIV-1 reverse transcriptase can reveal why particular mutations impart resistance to drugs such as streptomycin or AZT, but cannot yet be used to predict all mutations that will lead to drug resistance. In contrast, selection...