The interaction between the U1 snRNP‐specific U1 A protein and U1 snRNA has been analysed. The binding site for the protein on the RNA is shown to be in hairpin II, which extends from positions 48 to 91 in the RNA. Within this hairpin the evolutionarily conserved loop sequence is crucial for interaction with U1 A protein. U1 A protein can also bind the loop sequence when it is part of an artificial RNA which cannot form a stable hairpin structure. The region of the protein required to bind to U1 snRNA consists of a conserved 80 amino acid motif, previously identified in many ribonucleoprotein (RNP) proteins, together with (maximally) 11 N‐terminal and 10 C‐terminal flanking amino acids. Point mutations introduced into two of the most highly conserved regions of this motif abolish RNA binding. U1 snRNA mutants from which the U1 A binding site has been deleted are shown to be capable of assembly into RNP particles which are immunoprecipitable by patient antisera which recognize U1 A protein. The role of RNA‐protein and protein‐protein interactions in U snRNP assembly are discussed.

The basis of the specificity of interaction of U1 and U2 small nuclear (sn)RNAs and their cognate binding proteins, U1A and U2B'', has been examined. The U1A protein recognizes U1 snRNA on its own, whereas U2B'' binds specifically to U2 snRNA only in the presence of a second protein, U2A'. Exchange of two nucleotides between the two RNAs or of eight amino acids between the two proteins reverses binding specificity.

Defects in human DNA repair proteins can give rise to the autosomal recessive disorders xeroderma pigmentosum (XP) and Cockayne's syndrome (CS), sometimes even together. Seven XP and three CS complementation groups have been identified that are thought to be due to mutations in genes from the nucleotide excision repair pathway. Here we isolate frog and human complementary DNAs that encode proteins resembling RAD2, a protein involved in this pathway in yeast. Alignment of these three polypeptides, together with two other RAD2 related proteins, reveals that their conserved sequences are largely confined to two regions. Expression of the human cDNA in vivo restores to normal the sensitivity to ultraviolet light and unscheduled DNA synthesis of lymphoblastoid cells from XP group G, but not CS group A. The XP-G correcting protein XPGC is generated from a messenger RNA of approximately 4 kilobases that is present in normal amounts in the XP-G cell line.

The U2 snRNP contains two specific proteins, U2B″ and U2A'. Neither of these proteins, on its own, is capable of specific interactions with U2 RNA. Here, a complex between U2B″ and U2A' that forms in the absence of RNA is identified. Analysis of mutant forms of U2B″ shows that the smallest fragment able to bind specifically U2 RNA (amino acids 1‐88) is also the minimal region required for complex formation with U2A', and implies that this region must be largely structurally intact for U2A' interaction. Although this truncated U2B″ fragment is capable of making specific protein–RNA and protein‐protein interactions its structure, as measured by the ability to bind to U2A″, appears to depend on the rest of the protein. Hybrids between U2B″ and the closely related U1A protein are used to localize U2B″ specific amino acids involved in protein‐protein interaction. These can be divided into two functional groups. U2A' interaction with U2B″ amino acids 37‐46 permits binding to U2 RNA whereas interaction with U2B″ specific amino acids between positions 14 and 25 reduces non‐specific binding to U1 RNA. These two proteins may serve as a general example of how RNA binding may be modulated by protein‐protein interaction in the assembly of RNPs, particularly since the region of U2″ involved in interaction with U2A' consists mainly of a conserved RNP motif.

No abstract

SummaryWearable devices are fast evolving to address mobility and autonomy needs of elderly people who would benefit from physical assistance. Recent developments in soft robotics provide important opportunities to develop soft exoskeletons (also called exosuits) to enable both physical assistance and improved usability and acceptance for users. The XoSoft EU project has developed a modular soft lower limb exoskeleton to assist people with low mobility impairments. In this paper, we present the design of a soft modular lower limb exoskeleton to improve person’s mobility, contributing to independence and enhancing quality of life. The novelty of this work is the integration of quasi-passive elements in a soft exoskeleton. The exoskeleton provides mechanical assistance for subjects with low mobility impairments reducing energy requirements between 10% and 20%. Investigation of different control strategies based on gait segmentation and actuation elements is presented. A first hip–knee unilateral prototype is described, developed, and its performance assessed on a post-stroke patient for straight walking. The study presents an analysis of the human–exoskeleton energy patterns by way of the task-based biological power generation. The resultant assistance, in terms of power, was 10.9% ± 2.2% for hip actuation and 9.3% ± 3.5% for knee actuation. The control strategy improved the gait and postural patterns by increasing joint angles and foot clearance at specific phases of the walking cycle.

Domains of U1 snRNA which are functionally important have been identified using a splicing complementation assay in Xenopus oocytes. Mutations in, and deletions of, all three of the hairpin loop structures near the 5′ end of the RNA are strongly deleterious. Similarly, mutation of the Sm binding site abolishes complementation activity. Analysis of the protein binding properties of the mutant U1 snRNAs reveals that three of the functionally important domains, the first two hairpin loops and the Sm binding site, are required for interaction with U1 snRNP proteins. The fourth functionally important domain does not detectably affect snRNP protein binding and is not evolutionarily conserved. All of the deleterious mutations are shown to have similar effects on in vivo splicing complex formation.

The U2 snRNP complex contains two specific proteins, U2B" and U2A'. We have analysed the interaction of U2A' with U2B" and with U2 RNA. U2A' can form an weak but detectable RNA-protein complex with U2 RNA and a stable protein complex with U2B". This protein-protein complex binds efficiently and specifically to U2 RNA. Binding experiments with mutant forms of U2A' shows that the region of U2A' essential for binding to U2B" is extensive, being located between amino acid position 1-164. The behaviour of the wild type U2A' protein, and in particular of a mutant version of the protein in which amino acids 3, 4 and 5 are mutated, suggests that U2A' forms a weak interaction with U2 RNA which helps to stabilize the U2A'-U2B"-U2 RNA complex. Mutants of U2 RNA were used to localize the region of U2 RNA important for interaction with U2A'. The results show that U2A' interacts with the stem of hairpin IV.