G.U base pairing motifs in ribosomal RNA.

An increasing number of recognition mechanisms in RNA are found to involve G.U base pairs. In order to detect new functional sites of this type, we exhaustively analyzed the sequence alignments and secondary structures of eubacterial and chloroplast 16S and 23S rRNA, seeking positions with high levels of G.U pairs. Approximately 120 such sites were identified and classified according to their secondary structure and sequence environment. Overall biases in the distribution of G.U pairs are consistent with previously proposed structural rules: the side of the wobble pair that is subject to a loss of stacking is preferentially exposed to a secondary structure loop, where stacking is not as essential as in helical regions. However, multiple sites violate these rules and display highly conserved G.U pairs in orientations that could cause severe stacking problems. In addition, three motifs displaying a conserved G.U pair in a specific sequence/structure environment occur at an unusually high frequency. These motifs, of which two had not been reported before, involve sequences 5''UG3'' 3''GA5'' and 5''UG3'' 3''GU5'', as well as G.U pairs flanked by a bulge loop 3'' of U. The possible structures and functions of these recurrent motifs are discussed.     

Two binding partners cooperate to activate the molecular motor Kinesin-1

The regulation of molecular motors is an important cellular problem, as motility in the absence of cargo results in futile adenosine triphosphate hydrolysis. When not transporting cargo, the microtubule (MT)-based motor Kinesin-1 is kept inactive as a result of a folded conformation that allows autoinhibition of the N-terminal motor by the C-terminal tail. The simplest model of Kinesin-1 activation posits that cargo binding to nonmotor regions relieves autoinhibition. In this study, we show that binding of the c-Jun N-terminal kinase-interacting protein 1 (JIP1) cargo protein is not sufficient to activate Kinesin-1. Because two regions of the Kinesin-1 tail are required for autoinhibition, we searched for a second molecule that contributes to activation of the motor. We identified fasciculation and elongation protein zeta1 (FEZ1) as a binding partner of kinesin heavy chain. We show that binding of JIP1 and FEZ1 to Kinesin-1 is sufficient to activate the motor for MT binding and motility. These results provide the first demonstration of the activation of a MT-based motor by cellular binding partners.     

Viral RNA pseudoknots: versatile motifs in gene expression and replication

RNA pseudoknots are structural elements found in almost all classes of RNA. First recognized in the genomes of plant viruses, they are now established as a widespread motif with diverse functions in various biological processes. This Review focuses on viral pseudoknots and their role in virus gene expression and genome replication. Although emphasis is placed on those well defined pseudoknots that are involved in unusual mechanisms of viral translational initiation and elongation, the broader roles of pseudoknots are also discussed, including comparisons with relevant cellular counterparts. The relationship between RNA pseudoknot structure and function is also addressed.     

Combination of RNA interference and U1 inhibition leads to increased inhibition of gene expression

RNA interference (RNAi) has been revolutionary for the specific inhibition of gene expression. However, the application of RNAi has been hampered by the fact that many siRNAs induce dose-dependent unwanted secondary effects. Therefore, new methods to increase inhibition of gene expression with low doses of inhibitors are required. We have tested the combination of RNAi and U1i (U1 small nuclear RNA—snRNA—interference). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to target a pre-mRNA and inhibit its gene expression by blocking nuclear polyadenylation. The combination of RNAi and U1i resulted in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. The increased inhibition observed is stable over time and allows higher inhibition than the best obtained with either of the inhibitors alone even with decreased doses of the inhibitors. We believe that the combination of RNAi and U1i will be of interest when higher inhibition is required or when potent inhibitors are not available. Also, the combination of these techniques would allow functional inhibition with a decreased dose of inhibitors, avoiding toxicity due to dose-dependent unwanted effects.     

Structure and expression of a chicken gene coding for U1 RNA

We have isolated and sequenced a genomic fragment containing sequences complementary to chicken U1 RNA. The sequence of this genomic U1 gene is completely homologous and colinear with that of chicken U1 RNA. This U1 gene is part of a multigene family (6--10 copies per haploid genome), and these loci do not appear to be closely clustered. Sequences complementary to other snRNAs are not present within the 2.5 kb genomic fragment containing the U1 gene. We have determined that U1 RNA is synthesized by polymerase II; however, a "Hogness box" is not present upstream from its cap site at the position usually observed for mRNA genes. The synthesis of U1 RNA in oviduct nuclei during different states of hormonal induction also appears to be constitutive.     

A candidate gene for human U1 RNA.

Clones containing sequences complementary to the small nuclear RNA U1 were isolated from the human DNA library of Lawn et al. (1978). Three clones were studied by hybridization and restriction enzyme cleavage. The results showed that the inserts in all three clones were different and that each clone contains one single copy of a sequence which hybridizes to U1 RNA. The results revealed moreover that only one of the three clones contains all the cleavage sites which can be predicted from the known sequence of human U1 RNA, suggesting that the three clones comprise one candidate U1 gene and two pseudogenes. A fragment from the recombinant with the candidate U1 gene was subcloned in the pPR322 plasmid and part of its sequence was determined. The results showed that the subclone contains a sequence which matches that of the human U1 RNA perfectly. The sequence "TATAT" which often is found adjacent to RNA polymerase II start sites, was identified 33-37 base pairs upstream from the beginning of the U1 sequence. Two ten base pairs long, nearly perfect, direct repeats were also identified in the vicinity of the U1 sequence and an imperfect inverted repeat follows immediately after the U1 gene.