Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (aw) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650–0.605 aw. Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 aw). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 aw for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (∼0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 aw for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) are essential components of the nuclear pre-mRNA processing machinery. A hallmark of these particles is a ring-shaped core domain generated by the binding of Sm proteins onto snRNA. PRMT5 and SMN complexes mediate the formation of the core domain in vivo. Here, we have elucidated the mechanism of this reaction by both biochemical and structural studies. We show that pICln, a component of the PRMT5 complex, induces the formation of an otherwise unstable higher-order Sm protein unit. In this state, the Sm proteins are kinetically trapped, preventing their association with snRNA. The SMN complex subsequently binds to these Sm protein units, dissociates pICln, and catalyzes ring closure on snRNA. Our data identify pICln as an assembly chaperone and the SMN complex as a catalyst of spliceosomal snRNP formation. The mode of action of this combined chaperone/catalyst system is reminiscent of the mechanism employed by DNA clamp loaders.

Despite extensive studies on mammalian neurogenesis, its post-transcriptional regulation remains under-explored. Here we report that neural-specific inactivation of two murine post-transcriptional regulators, Pumilio 1 (Pum1) and Pum2, severely reduced the number of neural stem cells (NSCs) in the postnatal dentate gyrus (DG), drastically increased perinatal apoptosis, altered DG cell composition, and impaired learning and memory. Consistently, the mutant DG neurospheres generated fewer NSCs with defects in proliferation, survival, and differentiation, supporting a major role of Pum1 and Pum2 in hippocampal neurogenesis and function. Cross-linking immunoprecipitation revealed that Pum1 and Pum2 bind to thousands of mRNAs, with at least 694 common targets in multiple neurogenic pathways. Depleting Pum1 and/or Pum2 did not change the abundance of most target mRNAs but up-regulated their proteins, indicating that Pum1 and Pum2 regulate the translation of their target mRNAs. Moreover, Pum1 and Pum2 display RNA-dependent interaction with fragile X mental retardation protein (FMRP) and bind to one another's mRNA. This indicates that Pum proteins might form collaborative networks with FMRP and possibly other post-transcriptional regulators to regulate neurogenesis.

Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs.Several nuclear RNA-protein complexes (RNPs) 3 involved in the processing of mRNAs, such as the snRNPs of the major (U1, U2, U4/6, and U5) and minor (U11, U12, U5, and U4/6atac) spliceosome and the histone-mRNA processing U7 snRNP contain a set of evolutionary conserved proteins of the Sm/LSm class (1, 2). This group of proteins has the propensity to form heptameric rings in the presence of their respective target snRNA. Sm and LSm/Sm rings (also called "cores") can form spontaneously in vitro on their target RNAs (3-5). However, assembly in vivo occurs in a highly regulated manner and is assisted by trans-acting factors. One well characterized entity in this pathway is the SMN complex, whose name-giving component SMN is the product of the spinal muscular atrophy (SMA) disease gene (6, 7). This entity recruits all Sm proteins and promotes their transfer onto the U snRNAs (8 -10). Likewise, assembly of the U7 snRNP is facilitated by a specialized SMN complex that is charged with the unique set of Sm and LSm proteins of this particle (11). With a sedimentation coefficient of 25-40 S and an estimated molecular mass exceeding 1 megadalton, the SMN complex represents a macromolecular machine of great complexity. So far, nine major proteins termed SMN, Gemins 2-8, and unrip as well as nine Sm/LSm protein "substrates" (i.e. B/BЈ, D1, D2, D3, E, F, G, LSm10, and LSm11) have been identified as components of this assembly machinery (12, 13).Our knowledge about the architecture of the core SM...

Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, referred to as Polycomb domains. Non-canonical PRC1 complexes retain RING1/RNF2 E3-ubiquitin ligases but have unique sets of accessory subunits. How these non-canonical complexes recognize and regulate their gene targets remains poorly understood. Here, we show that the BCL6 co-repressor (BCOR), a member of the PRC1.1 complex, is critical for maintaining primed pluripotency in human embryonic stem cells (ESCs). BCOR depletion leads to the erosion of Polycomb domains at key developmental loci and the initiation of differentiation along endoderm and mesoderm lineages. The C terminus of BCOR regulates the assembly and targeting of the PRC1.1 complex, while the N terminus contributes to BCOR-PRC1.1 repressor function. Our findings advance understanding of Polycomb targeting and repression in ESCs and could apply broadly across developmental systems.

The assembly of the Sm-class of uridine-rich small nuclear ribonucleoproteins (U snRNPs), albeit spontaneous in vitro, has recently been shown to be dependent on the aid of a large number of assisting factors in vivo. These factors are organized in two interacting units termed survival motor neuron (SMN)-and protein arginine methyltransferase 5 (PRMT5)-complexes, respectively. While the PRMT5-complex acts early in the assembly pathway by activating common proteins of U snRNPs, the SMN-complex functions to join proteins and RNA in a highly ordered, apparently regulated manner. Here, we summarize recent progress in the understanding of this process and discuss the influence exerted by the aforementioned trans-acting factors. The ability to reconstitute macromolecular complexes from purified single components in vitro has fostered the concept of self-assembly, one of the central principles of molecular biology. From a theoretical, ''in vitro'' point of view, the formation of macromolecular complexes depends on diffusiondriven, random, and reversible encounters of the cognate subunits [1,2]. The stability of the resulting entities is determined by the ratio of the respective association and dissociation rate constants. While these considerations hold true for idealized, aqueous solutions, the situation in vivo is markedly different. In cells, the local concentration of individual components of macromolecular complexes and other proteins is usually relatively low, yet as a whole they occupy a significant fraction of the total volume. Hence, the possibility for unspecific interactions arises, which hinder the assembly pathway. Individual molecules therefore need to be directed to the site of complex assembly, in order to increase their local concentration and protect them against unfavorable interactions. Furthermore, a separation of both the site of assembly and the site of function should help to prevent assembly intermediates from adversely affecting the function of fully assembled macromolecular complexes. Taking these considerations into account, it is not surprising that cells have evolved strategies to ensure the faithful generation of macromolecular assemblies [1]. Among others, three features seem to predominate: (1) the segregation of biosynthesis of individual components and their assembly into higher-order structures into different subcellular compartments; (2) the evolution of molecular chaperones, which promote formation of intermediates, shielding these intermediates from adverse, premature interactions with substrate molecules of the finally assembled macromolecular complexes; and (3) trans-acting factors, working as scaffolds to coordinate several processes of the assembly reaction. A number of biological processes follow, at least in part, these principles and well known examples are the assembly of proteasomes and nucleosomes [3,4].In this review, we summarize recent experimental advances in the understanding of the in vivo biogenesis pathway of macromolecular RNA-protein complexes found in...

The assembly of spliceosomal U snRNPs depends on the coordinated action of PRMT5 and SMN complexes in vivo. These trans-acting factors enable the faithful delivery of seven Sm proteins onto snRNA and the formation of the common core of snRNPs. To gain mechanistic insight into their mode of action, we reconstituted the assembly machinery from recombinant sources. We uncover a stepwise and ordered formation of distinct Sm protein complexes on the PRMT5 complex, which is facilitated by the assembly chaperone pICln. Upon completion, the formed pIClnSm units are displaced by new pICln-Sm protein substrates and transferred onto the SMN complex. The latter acts as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to prevent mis-assembly and to ensure the transfer of Sm proteins to cognate RNA. Investigation of mutant SMN complexes provided insight into the contribution of individual proteins to these activities. The biochemical reconstitution presented here provides a basis for a detailed molecular dissection of the U snRNP assembly reaction.

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