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U7 snRNPs were isolated from HeLa cells by biochemical fractionation, followed by affinity purification with a biotinylated oligonucleotide complementary to U7 snRNA. Purified U7 snRNPs lack the Sm proteins D1 and D2, but contain additional polypeptides of 14, 50 and 70 kDa. Microsequencing identified the 14 kDa polypeptide as a new Sm-like protein related to Sm D1 and D3. Like U7 snRNA, this protein, named Lsm10, is enriched in Cajal bodies of the cell nucleus. Its incorporation into U7 snRNPs is largely dictated by the special Sm binding site of U7 snRNA. This novel type of Sm complex, composed of both conventional Sm proteins and the Sm-like Lsm10, is most likely to be important for U7 snRNP function and subcellular localization.

The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S. cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene.

These observations implicate EIF4E, and more specifically control of EIF4E activity, directly in autism. The findings raise the exciting possibility that pharmacological manipulation of EIF4E may provide therapeutic benefit for those with autism caused by disturbance of the converging pathways controlling EIF4E activity.

The SOS-inducible ruvA and ruvB gene products ofEscherichia coli are required for normal levels ofgenetic recombination and DNA repair. In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA. This process, known as branch migration, is important for the formation of heteroduplex DNA. In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA. Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5' -b 3' polarity. The reaction is ATPdependent and its efficiency is inversely related to the length of the duplex DNA. These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.DNA helicases play essential roles in DNA replication, repair, and recombination (for review see ref. 1). In bacteria, helicases such as Rep, DnaB, and PriA (n') act at the replication fork, where they unwind DNA during replication (2). Unwinding occurs with a defined polarity and is driven at the expense of nucleoside triphosphate hydrolysis. In DNA repair, the UvrA and UvrB proteins, part of the UvrABC excision nuclease complex, exhibit helicase activity during the recognition of DNA lesions (3), while UvrD (DNA helicase II) is involved in the disassembly of post-incision complexes (4).During genetic recombination in Escherichia coli, the formation of recombinant DNA molecules occurs via a series of well-defined, yet overlapping steps, several of which involve the action of DNA helicases. For example, RecBCD enzyme unwinds duplex DNA leading to the initiation of recombination by RecA protein (5, 6). A similar role is likely to be played by the RecQ helicase (7). In early studies with RecA protein, it was thought that the mechanisms ofhomologous pairing and strand exchange might involve strand separation. However, this was not the case (8) and current work indicates the formation of multistranded DNA helices within the RecA filament (9-14). Nevertheless, the concept that subsequent branch migration of a Holliday junction and the formation of extensive lengths of heteroduplex DNA might be catalyzed by a helicase-like activity remains attractive.In recent studies we focused our attention on the proteins encoded by the ruv locus of the E. coli chromosome. The RuvA and RuvB proteins interact with each other and catalyze reactions that are important for genetic recombination and the recombinational repair of DNA damage. Early genetic studies showed that ruvA and ruvB mutants had similar phenotypes characteristic of a defect in a late step of recombination, such as the processing of recombination intermediates (15-17). Biochemical studies provided support for this notion by demonstrating that RuvA and RuvB together promote the branch migration of Holliday junctions in vitro, leading to the formation of heteroduplex DNA (...

BackgroundMutation in the UPF3B gene on chromosome X is implicated in neurodevelopmental disorders including X-linked intellectual disability, autism and schizophrenia. The protein UPF3B is involved in the nonsense-mediated mRNA decay pathway (NMD) that controls mRNA stability and functions in the prevention of the synthesis of truncated proteins.ResultsHere we show that NMD pathway components UPF3B and UPF1 are down-regulated during differentiation of neural stem cells into neurons. Using tethered function assays we found that UPF3B missense mutations described in families with neurodevelopmental disorders reduced the activity of UPF3B protein in NMD. In neural stem cells, UPF3B protein was detected in the cytoplasm and in the nucleus. Similarly in neurons, UPF3B protein was detected in neurites, the somatic cytoplasm and in the nucleus. In both cell types nuclear UPF3B protein was enriched in the nucleolus. Using GFP tagged UPF3B proteins we found that the missense mutations did not affect the cellular localisation. Expression of missense mutant UPF3B disturbed neuronal differentiation and reduced the complexity of the branching of neurites. Neuronal differentiation was similarly affected in the presence of the NMD inhibitor Amlexanox. The expression of mutant UPF3B proteins lead to a subtle increase in mRNA levels of selected NMD targets.ConclusionsTogether our findings indicate that, despite the down-regulation of NMD factors, functional NMD is critical for neuronal differentiation. We propose that the neurodevelopmental phenotype of UPF3B missense mutation is caused by impairment of NMD function altering neuronal differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0122-1) contains supplementary material, which is available to authorized users.

Metazoan replication-dependent histone mRNAs do not have a poly(A) tail but end instead in a conserved stem-loop structure. Efficient translation of these mRNAs is dependent on the stem-loop binding protein (SLBP). Here we explore the mechanism by which SLBP stimulates translation in vertebrate cells, using the tethered function assay and analyzing protein-protein interactions. We show for the first time that translational stimulation by SLBP increases during oocyte maturation and that SLBP stimulates translation at the level of initiation. We demonstrate that SLBP can interact directly with subunit h of eIF3 and with Paip1; however, neither of these interactions is sufficient to mediate its effects on translation. We find that Xenopus SLBP1 functions primarily at an early stage in the cap-dependent initiation pathway, targeting small ribosomal subunit recruitment. Analysis of IRES-driven translation in Xenopus oocytes suggests that SLBP activity requires eIF4E. We propose a model in which a novel factor contacts eIF4E bound to the 5 0 cap and SLBP bound to the 3 0 end simultaneously, mediating formation of an alternative end-to-end complex.

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