Wilbert C. Boelens

The HIV-1 Rev Activation Domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs

HIV-1 Rev protein directs nuclear export of pre-mRNAs and mRNAs containing its binding site, the Rev response element (RRE). To define how Rev acts, we used conjugates between bovine serum albumin (BSA) and peptides comprising the Rev activation domain (BSA-R). BSA-R inhibited Rev-mediated nuclear RNA export, whereas a mutant activation domain peptide conjugate did not. BSA-R did not affect the export of mRNA, tRNA, or ribosomal subunits, but did inhibit export of 5S rRNA and spliceosomal U snRNAs. BSA-R was itself exported from the nucleus in an active, saturable manner. Thus, the Rev activation domain constitutes a nuclear export signal that redirects RRE-containing viral RNAs to a non-mRNA export pathway.

Murine leukemia virus-induced T-cell lymphomagenesis: Integration of proviruses in a distinct chromosomal region

A number of mink cell focus-forming (MCF) proviruses was molecularly cloned from mouse lymphoma DNA. From each clone, flanking probes were prepared to detect common integration regions in other MuLV-induced lymphomas. One clone frequently revealed variations in the molecular structure of the corresponding region (Pim-1) in other lymphomas. The results show the following. Changes in the Pim region are seen in 24 out of 93 lymphomas tested. Over 50% of the early T-cell lymphomas show integration in the Pim-1 region. The alterations are seen in different mouse strains and with various MuLVs. The observed variations are caused by the integration of predominantly MCF genomes. All integrations occur in a region spanning less than 20 kb and are associated with the transcriptional activation of a distinct region within the Pim-1 domain. The activated region does not show any homology with 13 known and three putative oncogenes.

The human genome encodes 10 α-crystallin–related small heat shock proteins: HspB1–10

Abstract To obtain an inventory of all human genes that code for α-crystallin–related small heat shock proteins (sHsps), the databases available from the public International Human Genome Sequencing Consortium (IHGSC) and the private Celera human genome project were exhaustively searched. Using the human Hsp27 protein sequence as a query in the protein databases, which are derived from the predicted genes in the human genome, 10 sHsp-like proteins were retrieved, including Hsp27 itself. Repeating the search procedure with all 10 proteins and with a variety of more distantly related animal sHsps, no further human sHsps were detected, as was the case when searches were performed at deoxyribonucleic acid level. The 10 retrieved proteins comprised the 9 earlier recognized human sHsps (Hsp27/HspB1, HspB2, HspB3, αA-crystallin/HspB4, αB-crystallin/HspB5, Hsp20/HspB6, cvHsp/HspB7, H11/HspB8, and HspB9) and a sperm tail protein known since 1993 as outer dense fiber protein 1 (ODF1). Although this latter protein probably serves a structural role and has a high cysteine content (14%), it clearly contains an α-crystallin domain that is characteristic for sHsps. ODF1 can as such be designated as HspB10. The expression of all 10 human sHsp genes was confirmed by expressed sequence tag (EST) searches. For Hsp27/HspB1, 2 retropseudogenes were detected. The HspB1–10 genes are dispersed over 9 chromosomes, reflecting their ancient origin. Two of the genes (HspB3 and HspB9) are intronless, and the others have 1 or 2 introns at various positions. The transcripts of several sHsp genes, notably HspB7, display low levels of alternative splicing, as supported by EST evidence, which may result in minor amounts of isoforms at the protein level.

Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA.

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.

Crystal Structures of α-Crystallin Domain Dimers of αB-Crystallin and Hsp20

Small heat shock proteins (sHsps) are a family of large and dynamic oligomers highly expressed in long-lived cells of muscle, lens and brain. Several family members are upregulated during stress, and some are strongly cytoprotective. Their polydispersity has hindered high-resolution structure analyses, particularly for vertebrate sHsps. Here, crystal structures of excised alpha-crystallin domain from rat Hsp20 and that from human alphaB-crystallin show that they form homodimers with a shared groove at the interface by extending a beta sheet. However, the two dimers differ in the register of their interfaces. The dimers have empty pockets that in large assemblies will likely be filled by hydrophobic sequence motifs from partner chains. In the Hsp20 dimer, the shared groove is partially filled by peptide in polyproline II conformation. Structural homology with other sHsp crystal structures indicates that in full-length chains the groove is likely filled by an N-terminal extension. Inside the groove is a symmetry-related functionally important arginine that is mutated, or its equivalent, in family members in a range of neuromuscular diseases and cataract. Analyses of residues within the groove of the alphaB-crystallin interface show that it has a high density of positive charges. The disease mutant R120G alpha-crystallin domain dimer was found to be more stable at acidic pH, suggesting that the mutation affects the normal dynamics of sHsp assembly. The structures provide a starting point for modelling higher assembly by defining the spatial locations of grooves and pockets in a basic dimeric assembly unit. The structures provide a high-resolution view of a candidate functional state of an sHsp that could bind non-native client proteins or specific components from cytoprotective pathways. The empty pockets and groove provide a starting model for designing drugs to inhibit those sHsps that have a negative effect on cancer treatment.

The human U1A snRNP protein regulates polyadenylation via a direct interaction with poly(A) polymerase

The human U1 snRNP-specific U1A protein autoregulates its production by binding its own pre-mRNA and inhibiting polyadenylation. The mechanism of this regulation has been elucidated by in vitro studies. U1A protein is shown not to prevent either binding of cleavage and polyadenylation specificity factor (CPSF) to its recognition sequence (AUUAAA) or to prevent cleavage of U1A pre-mRNA. Instead, U1A protein bound to U1A pre-mRNA inhibits both specific and nonspecific polyadenylation by mammalian, but not by yeast, poly(A) polymerase (PAP). Domains are identified in both proteins whose removal uncouples the polyadenylation activity of mammalian PAP from its inhibition via RNA-bound U1A protein. Finally, U1A protein is shown to specifically interact with mammalian PAP in vitro. The possibility that this interaction may reflect a broader role of the U1A protein in polyadenylation is discussed.

Autoantibodies to cytosolic 5′-nucleotidase 1A in inclusion body myositis

Sporadic inclusion body myositis (sIBM) is an inflammatory myopathy characterized by both degenerative and autoimmune features. In contrast to other inflammatory myopathies, myositis‐specific autoantibodies had not been found in sIBM patients until recently. We used human skeletal muscle extracts as a source of antigens to detect autoantibodies in sIBM and to characterize the corresponding antigen.

Nup145p is required for nuclear export of mRNA and binds homopolymeric RNA in vitro via a novel conserved motif

An essential yeast protein, Nup145p, is identified via its genetic interaction with the nucleoporin Nsp1p. Nup145p contains GLFG repeats and localizes to nuclear pores. Depletion of Nup145p in vivo leads rapidly to nuclear retention of polyadenylated RNAs and more slowly to cytoplasmic accumulation of a nuclear reporter protein. A stretch of 140 amino acids within Nup145p is conserved in two other yeast nucleoporins, Nup116p and Nup100p, and in an uncharacterized C. elegans protein. Genetic experiments in yeast reveal that the three copies of the motif carry out an essential, redundant function. Fragments of Nup145p and Nup116p including this motif bind specifically to homopolymeric RNAs in vitro. Nup145p, Nup116p, and Nup100p thus represent a novel class of nucleoporins involved in nucleocytoplasmic transport.

Preventing thiol-yne addition improves the specificity of strain-promoted azide-alkyne cycloaddition

The 1,3-dipolar cycloaddition of azides with ring-strained alkynes is one of the few bioorthogonal reactions suitable for specific biomolecule labeling in complex biological systems. Nevertheless, azide-independent labeling of proteins by strained alkynes can occur to a varying extent, thereby limiting the sensitivity of assays based on strain-promoted azide-alkyne cycloaddition (SPAAC). In this study, a subset of three cyclooctynes, dibenzocyclooctyne (DIBO), azadibenzocyclooctyne (DIBAC), and bicyclo[6.1.0]nonyne (BCN), was used to evaluate the azide-independent labeling of proteins in vitro. For all three cyclooctynes, we show that thiol-yne addition with reduced peptidylcysteines is responsible for most of the azide-independent polypeptide labeling. The identity of the reaction product was confirmed by LC-MS and NMR analysis. Moreover, we show that undesired thiol-yne reactions can be prevented by alkylating peptidylcysteine thiols with iodoacetamide (IAM). Since IAM is compatible with SPAAC, a more specific azide-dependent labeling is achieved by preincubating proteins containing reduced cysteines with IAM.

The Small Heat-shock Protein αB-Crystallin Promotes FBX4-dependent Ubiquitination

αB-Crystallin is a small heat-shock protein in which three serine residues (positions 19, 45, and 59) can be phosphorylated under various conditions. We describe here the interaction of αB-crystallin with FBX4, an F-box-containing protein that is a component of the ubiquitin-protein isopeptide ligase SCF (SKP1/CUL1/F-box). The interaction with FBX4 was enhanced by mimicking phosphorylation of αB-crystallin at both Ser-19 and Ser-45 (S19D/S45D), but not at other combinations. Ser-19 and Ser-45 are preferentially phosphorylated during the mitotic phase of the cell cycle. Also αB-crystallin R120G, a mutant found to co-segregate with a desmin-related myopathy, displayed increased interaction with FBX4. Both αB-crystallin S19D/S45D and R120G specifically translocated FBX4 to the detergent-insoluble fraction and stimulated the ubiquitination of one or a few yet unknown proteins. These findings implicate the involvement of αB-crystallin in the ubiquitin/proteasome pathway in a phosphorylation- and cell cycle-dependent manner and may provide new insights into the αB-crystallin-induced aggregation in desmin-related myopathy.