Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly

It has been widely accepted that the early spliceosome assembly begins with U1 small nuclear ribonucleoprotein (U1 snRNP) binding to the 5' splice site (5'SS), which is assisted by the Ser/Arg (SR)-rich proteins in mammalian cells. In this process, the RS domain of SR proteins is thought to directly interact with the RS motif of U1-70K, which is subject to regulation by RS domain phosphorylation. Here we report that the early spliceosome assembly event is mediated by the RNA recognition domains (RRM) of serine/arginine-rich splicing factor 1 (SRSF1), which bridges the RRM of U1-70K to pre-mRNA by using the surface opposite to the RNA binding site. Specific mutation in the RRM of SRSF1 that disrupted the RRM-RRM interaction also inhibits the formation of spliceosomal E complex and splicing. We further demonstrate that the hypo-phosphorylated RS domain of SRSF1 interacts with its own RRM, thus competing with U1-70K binding, whereas the hyper-phosphorylated RS domain permits the formation of a ternary complex containing ESE, an SR protein, and U1 snRNP. Therefore, phosphorylation of the RS domain in SRSF1 appears to induce a key molecular switch from intra- to intermolecular interactions, suggesting a plausible mechanism for the documented requirement for the phosphorylation/dephosphorylation cycle during pre-mRNA splicing.     

Rest-activity circadian rhythms in aged Nothobranchius korthausae. The effects of melatonin

Adult (48-week-old) and senescent (72-week-old) individually-kept Nothobranchius korthausae were used as experimental subjects to characterise circadian system (CS) function and age-related changes in senescent fish. This species was specifically chosen because it has already shown potential for use as a model system in gerontological studies. The rest-activity rhythm (RAR) in fish can be easily monitored and used to characterise the state of the CS, and it has also been proposed as a reliable model to study sleep-like periods in fish. As they aged, N. korthausae experienced a significant decrease in total daily activity and a progressive impairment of the RAR, accompanied by changes in the regularity, fragmentation and amplitude of the rhythm. The ability of the CS to oscillate autonomously when the two main synchronizers, photoperiod and feeding time, were absent (continuous darkness and random feeding), was also impaired with age, as the capacity to re-synchronise to the light–dark (LD) cycle declined. Melatonin treatment improved the regularity, fragmentation and amplitude of the RAR in senescent fish, and it also improved sleep efficiency. In conclusion, N. korthausae represents a viable model for studying the aging of the circadian system and the restorative effect of chronobiotic substances, such as melatonin.     

Drosophila circadian rhythms in seminatural environments: Summer afternoon component is not an artifact and requires TrpA1 channels

Under standard laboratory conditions of rectangular light/dark cycles and constant warm temperature, Drosophila melanogaster show bursts of morning (M) and evening (E) locomotor activity and a “siesta” in the middle of the day. These M and E components have been critical for developing the neuronal dual oscillator model in which clock gene expression in key cells generates the circadian phenotype. However, under natural European summer conditions of cycling temperature and light intensity, an additional prominent afternoon (A) component that replaces the siesta is observed. This component has been described as an “artifact” of the TriKinetics locomotor monitoring system that is used by many circadian laboratories world wide. Using video recordings, we show that the A component is not an artifact, neither in the glass tubes used in TriKinetics monitors nor in open-field arenas. By studying various mutants in the visual and peripheral and internal thermo-sensitive pathways, we reveal that the M component is predominantly dependent on visual input, whereas the A component requires the internal thermo-sensitive channel transient receptor potential A1 (TrpA1). Knockdown of TrpA1 in different neuronal groups reveals that the reported expression of TrpA1 in clock neurons is unlikely to be involved in generating the summer locomotor profile, suggesting that other TrpA1 neurons are responsible for the A component. Studies of circadian rhythms under seminatural conditions therefore provide additional insights into the molecular basis of circadian entrainment that would otherwise be lost under the usual standard laboratory protocols.The circadian clock infiltrates almost every aspect of behavior and physiology of higher organisms and even some bacteria. Most studies of 24-h rhythms are carried out under strictly controlled laboratory conditions, an approach leading to a remarkably informative dissection of the clock, whose main molecular cogs are conserved among vertebrates and insects. Laboratory experiments are often extrapolated to the wild with the assumption that they reflect the natural situation. However, recent seminatural studies in mice, hamsters, and Drosophila have revealed some unexpected findings. For example, the widely held belief from laboratory studies that mice and golden hamsters are nocturnal needs to be revised because in the wild they are predominantly or exclusively diurnal (1, 2). Similarly in Drosophila melanogaster, locomotor rhythms studied in seminatural conditions reveal that deeply held, laboratory-derived assumptions may require significant revision. These include the crepuscular nature of fly activity, the role of the clock in “morning anticipation” and midday “siesta,” the requirement for clock gene expression in the central clock neurons for entrainment, and the role of light/dark (LD) cycles as the most important environmental Zeitgeber (“time giver”) in entraining the clock (3).Vanin et al. (3) observed that in the wild the phase of various features of circadian locomotor behavior such as the morning (M) and evening (E) components was best predicted by temperature, rather than “anticipation” of dawn and dusk over the seasonal LD cycle. In addition, at the warmer temperatures of European summers, flies did not generate an afternoon (A) siesta as in the laboratory. Instead, they dramatically increased their activity so that the major component of their locomotor profile was now the newly described A peak. The phase of the A component was modulated by mutation at the period (per) locus, suggesting that A represented a clock-mediated escape response from heat-induced stress (3, 4). Most surprisingly, null mutants of the negative regulators of the circadian clock period (per⁰¹) and timeless (tim⁰¹) exhibited naturally entrained behavioral profiles largely indistinguishable from those of wild-type strains. In sharp contrast, under laboratory conditions of constant 25 °C temperature and rectangular LD cycles, per⁰¹ and tim⁰¹ flies show no anticipation of dawn/dusk, and these mutants simply react to light-on or light-off signals with startle effects (5). The anticipatory nature of the M and E components in the laboratory led directly to the development of the dual oscillator model in the fly in which the Pigment Dispersing Factor (PDF) expressing s-LNv and l-LNvs (small and large lateral ventral neurons) generate the M locomotor component, whereas the dorsal lateral neurons, LNds, and dorsal neurons, DNs, produce the E component (6, 7).Although Vanin et al. focused predominantly on the phases of the major locomotor components under natural lighting and thermal conditions (3), in a similar natural study, Menegazzi et al. suggested that although per null mutants look similar in their behavioral phasing to wild type, the A peak tends to be larger in per⁰¹ mutants (4). These authors suggested that PER normally serves to reduce the amount of “inappropriate” activity that occurs during the warmest part of the day (4). Although their results were based on a very small sample of flies on a few days of recording, they were nevertheless welcomed in that they revealed that possessing a wild-type clock appeared to be behaviorally adaptive compared with having a severely disturbed clock.Another study performed under seminatural conditions at tropical latitudes has questioned the validity of the A component (8). These authors suggest instead that A represents a behavioral artifact as a result of flies avoiding the midday sun by sheltering in the shaded part of the glass activity tube where the TriKinetics infrared detectors are located, leading to inappropriate triggering of the sensor and high activity counts. In apparent support of this model, they observed that flies in open-field Petri dish arenas did not show an A component under summer conditions, although this interpretation has been criticized (9), in part because Petri dishes are well known to be problematic for Drosophila open-field behavioral recordings (10).Given the interest generated by Vanin et al. (3), we have revisited these natural studies and extended them with more sophisticated simulations of natural temperature and light cycles in the laboratory. By using video recordings of fly circadian activity in glass tubes and open-field arenas, we investigate whether the A component is an artifact. Furthermore, in both the Vanin et al. (3) and the Menegazzi et al. (4) studies, the classic per mutants were congenic with each other but were compared with three different wild-type strains so genetic background was not controlled. Using congenic controls we reexamine whether we can observe a phenotype for arrhythmic mutants in simulated seminatural conditions. Finally we study the A peak in a range of photoreceptor and thermoreceptor mutants to investigate the underlying genetic and neuroanatomical basis for this newly identified summer element of circadian behavior.     

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function

We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation–contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation–contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing.The expression of more than 95% of human genes is affected by alternative pre-mRNA splicing (AS) (1, 2). Differentially spliced isoforms play distinct roles in a temporally and spatially specific manner (3), and mutations that lead to aberrant splicing are the cause of many human genetic diseases (4). RNA-binding proteins (RBPs) play a central role in the regulation of alternative splicing during development and disease. They function primarily by positively or negatively regulating splice-site recognition by the spliceosome (1). Many RBPs are expressed in specific tissues, and AS is regulated by the combinatorial activities of these factors on specific pre-mRNAs through their interactions with distinct regulatory sequences in pre-mRNA that function as splicing enhancers or silencers (5).The developing heart is one of the best studied systems where splicing changes occur during normal development, and mutations affecting specific splicing outcomes contribute to cardiomyopathy (6, 7). Although these mutations can either disrupt splicing elements or affect the expression of specific splicing factors, the latter mechanism is clearly responsible for the distinct splicing profiles at different developmental stages. For example, the dynamics of alternative splicing during postnatal heart development correlate with expression changes of many RBPs, including CUG-BP, Elav-like family member 1 (CELF1), Muscleblind-like 1 (MBNL1), and FOX proteins (8). Detailed biochemical studies have elucidated the mechanisms by which these splicing factors regulate splicing in a position- and context-dependent manner (9, 10). The function of other RBPs during heart development has also been studied. For example, two of the muscle-specific splicing factors, RBM20 and RBM24, play distinct roles in splicing regulation. RBM20 mainly acts as a splicing repressor, as its absence leads to multiple exon inclusion events in the heart. For example, the Titin gene is one of the major targets of RBM20 (11, 12). On the other hand, loss of RBM24 expression gives rise to many exon skipping events (13), implicating RBM24 as a splicing activator. Strikingly, there is little overlap between RBM20 and RBM24 splicing targets, suggesting that RBM20 and RBM24 are involved in regulating splicing of distinct groups of pre-mRNAs and there is little cross-talk between these two splicing factors.Distinct splicing activities have also been ascribed to general splicing factors (1). There are two major types of ubiquitously expressed RBPs: the heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine/arginine (SR)-rich proteins. hnRNPs and SR proteins are generally believed to play opposite roles in splicing: SR proteins promote the inclusion of exons during splicing, whereas hnRNP proteins repress inclusion (1). The function of certain SR proteins has been studied in the mouse heart through the conditional knockout approach. Srsf1 deletion in the heart leads to lethal dilated cardiomyopathy (DCM) (death occurs 6–8 wk after birth) (14). SRSF1 is required for the cardiac-specific splicing of Cypher (also called Ldb3) pre-mRNA, and the regulation of alternative splicing of calcium/calmodulin-dependent protein kinase II delta (Camk2d) and cardiac Troponin T (cTnT) during heart development. In particular, the persistent splicing of a neuronal isoform of Camk2d and its ability to enhance excitation and contraction coupling (ECC) activity in Srsf1 mutant cardiomyocytes have been proposed as a possible cause of the phenotype in mutant mice (14). Ablation of another SR protein, SRSF10 (SRp38), from the mouse also leads to heart defects (15). SRSF10 has been shown to regulate the splicing of Triadin, an important component of ECC machinery (15). Interestingly, conditional deletion of Srsf2 from the heart leads to DCM with little splicing misregulation but instead affects the expression of the calcium channel Ryr2 (16). It is striking that these SR proteins affect ECC activity in the heart by directly regulating the expression/splicing of distinct players in this machinery. Because these studies were conducted before the advent of next-generation RNA sequencing, only a few splicing targets specifically regulated by these SR proteins were identified. A more comprehensive study of the effects of deleting the genes encoding these proteins from the heart on the splicing program has not been reported.In contrast to SR proteins, specific functions of hnRNP proteins in cardiac pre-mRNA splicing have not been determined. In this report, we selectively inactivated the expression of one of the most abundant hnRNP proteins—hnRNP U—in the heart. We found that Hnrnpu mutant mice develop a lethal DCM phenotype, with death occurring around 2 wk after birth. There are multiple cardiac defects in mutant hearts accompanied by many splicing alterations. Some of these splicing targets are commonly regulated by hnRNP U and other SR and RBM proteins. We also identified many hnRNP U-specific splicing targets in the heart, including an ECC component Junctin. The protein product of the alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site in Hnrnpu mutant cells and could contribute to abnormal cardiac function. Our study also enables comparisons of the roles of different splicing factors in heart development and function.     

Influence of the pineal gland and circadian rhythms in circulating levels of thyroid hormones of male hamsters

Thyroxin (T4) and triiodothyronine (T3) were measured by radioimmunoassay in serum of hamsters sacrificed at 4-hr intervals throughout the daily light-dark cycle (14L/10D). Both T4 and T3 concentrations increased significantly during the L period of the daily cycle and decreased during the D period of the cycle; A.M. versus P.M. differences in free thyroxin indices (FTI) were also studied using the T4 and T3 uptake assays of Nuclear Medical Laboratories (Dallas, Texas). The free thyroxin index was significantly greater in serum samples of hamsters sacrificed at 7 P.M. than at 7 A.M. (lights on at 6:30 A.M.). Serum taken at 7 P.M. had less unsaturated binding sites than serum taken at 7 A.M. No significant A.M. versus P.M. differences in free thyroxin index were found in blind hamsters, although blind hamsters had significantly lower T4 and FTI than controls. Placing melatonin in the drinking water at a dose of 80 μg/ml did not significantly influence hormone levels. The greatest difference in hormone concentrations between control and blinded hamsters was found in P.M. samples. Blind hamsters had FTIs that were 48% of P.M. controls. Pinealectomy prevented the effects of blinding on T4 levels and FTIs.     

Chromosome-level assembly of Arabidopsis thaliana Ler reveals the extent of translocation and inversion polymorphisms

Resequencing or reference-based assemblies reveal large parts of the small-scale sequence variation. However, they typically fail to separate such local variation into colinear and rearranged variation, because they usually do not recover the complement of large-scale rearrangements, including transpositions and inversions. Besides the availability of hundreds of genomes of diverse Arabidopsis thaliana accessions, there is so far only one full-length assembled genome: the reference sequence. We have assembled 117 Mb of the A. thaliana Landsberg erecta (Ler) genome into five chromosome-equivalent sequences using a combination of short Illumina reads, long PacBio reads, and linkage information. Whole-genome comparison against the reference sequence revealed 564 transpositions and 47 inversions comprising ∼3.6 Mb, in addition to 4.1 Mb of nonreference sequence, mostly originating from duplications. Although rearranged regions are not different in local divergence from colinear regions, they are drastically depleted for meiotic recombination in heterozygotes. Using a 1.2-Mb inversion as an example, we show that such rearrangement-mediated reduction of meiotic recombination can lead to genetically isolated haplotypes in the worldwide population of A. thaliana. Moreover, we found 105 single-copy genes, which were only present in the reference sequence or the Ler assembly, and 334 single-copy orthologs, which showed an additional copy in only one of the genomes. To our knowledge, this work gives first insights into the degree and type of variation, which will be revealed once complete assemblies will replace resequencing or other reference-dependent methods.Landsberg erecta (Ler) is presumably the second-most-used strain of Arabidopsis thaliana after the reference accession Columbia (Col-0). It is broadly known as Ler-0, which is an abbreviation for its accession code La-1 and a mutation in the ERECTA gene. In 1957, George Rédei, at the University of Missouri–Columbia, irradiated La-1 samples, which were provided by Friedrich Laibach and were collected in Landsberg an der Warthe (now called Gorzów Wielkopolski), Poland, where Ler-0–related genotypes are still present (1). Some seeds of the original batch were irradiated with X-rays, resulting, among others, in the isolation of the erecta (er) mutant (2, 3). Will Feenstra received this mutant from George Rédei in 1959 because he was interested in its erect growth habit and introduced it as the standard strain in the Department of Genetics at Wageningen University. There, he started a mutant induction program that was later continued by Jaap van der Veen and Maarten Koornneef. Mutants from this program as well as parental lines of recombinant inbred lines were mainly distributed as Ler-0 lines to other laboratories, reflecting the increasing interest in A. thaliana. Some descendants of Ler-0 were later renamed to Ler-1 and -2 to identify genotypes used in different laboratories, but most likely all derived from the original mutant isolated by Rédei, and we will collectively refer to them as “Ler.”First comparative analyses of the Ler genome included cytogenetic studies using pachytene cells and in situ hybridization (4, 5), suggesting a large inversion on the short arm of chromosome 4, as well as differences between 5S rDNA clusters compared with the genome of Col-0 (4). The first large-scale analysis of the Ler genome sequence was published together with the Col-0 reference sequence in 2000 (6). Within 92 Mb of random shotgun dideoxy sequencing reads, 25,274 SNPs and 14,570 indels were identified. Although this was a severe underestimation (7–11), the authors already observed that many of the large indels contained entire active genes, half of which were found at different loci in the genome of Ler, whereas others were entirely absent (6).The advent of next-generation sequencing greatly expanded the knowledge of natural genetic diversity in A. thaliana (reviewed in refs. 12 and 13). However, genome-wide studies on gene absence/presence polymorphisms were not repeated, because short-read analyses focused on small-scale changes only. To resolve large variation, reference-guided assemblies (8, 9, 14) and structural variation-identifying tools (15) were introduced. However, such methods mostly reveal local differences, which do not include the complement of large-scale rearrangements including inversions or transpositions.So far, two de novo assemblies of Ler have been published. The first was based on Illumina short-read data and resulted in an assembly with an N50 of 198 kb, showing similar performance as a reference-guided assembly (8). The second was based on a set of previously released Pacific Bioscience’s single-molecule, real-time sequencing (PacBio) data (16) and assembled the genome into 38 contigs with an N50 of 11.2 Mb (17). This drastic improvement outlines the potential of long-read sequencing technologies such as PacBio sequencing (18) to overcome the limitations of short-read methods because long reads can span many of the repetitive regions, which are presumably the most common reason for assembly breaks in short-read assemblies. Alternatively, low-fold long-read sequencing could be combined with cheaper short-read sequencing, either by using the short reads for error-correcting the long reads (19) or for integrating long-read information into short-read assemblies or vice versa (20).Despite the unprecedented contiguity of this long-read assembly, both earlier studies focused on methodological aspects and did not perform any whole-genome comparisons of the Ler genome or gene annotations, and, as a consequence, comprehensive reports on large-scale rearrangements and nonreference genes are still sparse. We have generated an advanced de novo assembly of Ler consisting of 117 Mb arranged into five sequences representing the five chromosomes, which is based on a combination of short-read assembly, long-read-based gap closure, and scaffolding based on genetic maps. This chromosome-scale assembly and its comparison with the reference assembly revealed features that are typically not analyzed within next-generation sequencing assemblies, including the location of a polymorphic rDNA cluster and centromeric repeats, as well as the exact makeup of all large rearrangements including a 1.2-Mb inversion on the short arm of chromosome 4. This inversion suppresses meiotic recombination in Ler and Col-0 hybrids, and we show that this suppression introduced genetically isolated inversion haplotypes into the worldwide population of A. thaliana. De novo gene annotation revealed hundreds of copy-number polymorphisms as well as novel genes that are entirely absent in one or the other genome. Finally, we report on variation in different Ler genomes, suggesting that some Ler lines feature unexpected footprints of an additional mutagenesis event.     

Variation in sequence and organization of splicing regulatory elements in vertebrate genes

Although core mechanisms and machinery of premRNA splicing are conserved from yeast to human, the details of intron recognition often differ, even between closely related organisms. For example, genes from the pufferfish Fugu rubripes generally contain one or more introns that are not properly spliced in mouse cells. Exploiting available genome sequence data, a battery of sequence analysis techniques was used to reach several conclusions about the organization and evolution of splicing regulatory elements in vertebrate genes. The classical splice site and putative branch site signals are completely conserved across the vertebrates studied (human, mouse, pufferfish, and zebrafish), and exonic splicing enhancers also appear broadly conserved in vertebrates. However, another class of splicing regulatory elements, the intronic splicing enhancers, appears to differ substantially between mammals and fish, with G triples (GGG) very abundant in mammalian introns but comparatively rare in fish. Conversely, short repeats of AC and GT are predicted to function as intronic splicing enhancers in fish but are not enriched in mammalian introns. Consistent with this pattern, exonic splicing enhancer-binding SR proteins are highly conserved across all vertebrates, whereas heterogeneous nuclear ribonucleoproteins, which bind many intronic sequences, vary in domain structure and even presence/absence between mammals and fish. Exploiting differences in intronic sequence composition, a statistical model was developed to predict the splicing phenotype of Fugu introns in mammalian systems and was used to engineer the spliceability of a Fugu intron in human cells by insertion of specific sequences, thereby rescuing splicing in human cells.     

Real-time luminescence monitoring of cell-cycle and respiratory oscillations in yeast

The use of luciferase reporters has become a precise, noninvasive, high-throughput method for real-time monitoring of promoter activity in living cells, especially for rhythmic biological processes such as circadian rhythms. We developed a destabilized firefly luciferase as a reporter for rhythmic promoter activity in both the cell division and respiratory cycles of the budding yeast Saccharomyces cerevisiae in which real-time luminescence reporters have not been previously applied. The continuous output of light from luciferase reporters allowed us to explore the relationship between the cell division cycle and the yeast respiratory oscillation, including the observation of responses to chemicals that cause phase shifting of the respiratory oscillations. Destabilized firefly luciferase is a good reporter of cell cycle position in synchronized or partially synchronized yeast cultures, in both batch and continuous cultures. In addition, the oxygen dependence of luciferase can be used under certain conditions as a genetically encodable oxygen monitor. Finally, we use this reporter to show that there is a direct correlation between premature induction of cell division and phase resetting of the respiratory oscillation under the continuous culture conditions tested.     

Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved γ-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.In eukaryotes, centromeres play a critical role in accurate chromosome segregation and in the maintenance of genome integrity through their regulated assembly and the maintenance of their cohesion until anaphase. Centromeres consist each of a central core (1) characterized epigenetically by the recruitment of the histone H3 variant CENH3 (CENP-A in animals). Extensive studies are still ongoing to identify the regulatory factors for loading and maintenance of CENH3 at centromeres. In yeast, suppressor of chromosome missegregation protein 3 was identified as a specific chaperone for CENP-A loading (2). In animals, the Mis18 complex, including the CENH3 assembly factor Kinetochore Null 2 (KNL2; also called Mis18BP1), recruits the cell cycle-dependent maintenance and deposition factor of CENP-A, HJURP (Holliday junction recognition protein), to centromeres (3). Recently, two Mis18-complex components, Eic1 and Eic2, were identified in fission yeast (4). Whereas Eic1 promotes CENH3 loading and maintenance, Eic2 is recruited at centromeres independently of its association with Mis18. Together with CENH3, the conserved kinetochore assembly protein CENP-C participates in pericentromeric cohesin recruitment (5). The CENH3 loading machinery changed rapidly during evolution, and a CENH3 chaperone has not been identified in plants thus far. Moreover, nothing is known about a possibly conserved interplay between CENH3 loading and sister chromatid cohesion at centromeres. Recently, the plant homolog of KNL2 was proposed as an upstream component for CENH3 deposition at centromeres (6). Finally, the regulation of centromeric complex positioning at the nuclear envelope environment is still elusive in plants.

Table S1.

Gene accession numbers

ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria

Self-sustainable oscillation of KaiC phosphorylation has been reconstituted in vitro, demonstrating that this cycle is the basic time generator of the circadian clock of cyanobacteria. Here we show that the ATPase activity of KaiC satisfies the characteristics of the circadian oscillation, the period length, and the temperature compensation. KaiC possesses extremely weak but stable ATPase activity (15 molecules of ATP per day), and the addition of KaiA and KaiB makes the activity oscillate with a circadian period in vitro. The ATPase activity of KaiC is inherently temperature-invariant, suggesting that temperature compensation of the circadian period could be driven by this simple biochemical reaction. Moreover, the activities of wild-type KaiC and five period-mutant proteins are directly proportional to their in vivo circadian frequencies, indicating that the ATPase activity defines the circadian period. Thus, we propose that KaiC ATPase activity constitutes the most fundamental reaction underlying circadian periodicity in cyanobacteria.     

A PHD-polycomb repressive complex 2 triggers the epigenetic silencing of FLC during vernalization

Vernalization, the acceleration of flowering by winter, involves cold-induced epigenetic silencing of Arabidopsis FLC. This process has been shown to require conserved Polycomb Repressive Complex 2 (PRC2) components including the Su(z)12 homologue, VRN2, and two plant homeodomain (PHD) finger proteins, VRN5 and VIN3. However, the sequence of events leading to FLC repression was unclear. Here we show that, contrary to expectations, VRN2 associates throughout the FLC locus independently of cold. The vernalization-induced silencing is triggered by the cold-dependent association of the PHD finger protein VRN5 to a specific domain in FLC intron 1, and this association is dependent on the cold-induced PHD protein VIN3. In plants returned to warm conditions, VRN5 distribution changes, and it associates more broadly over FLC, coincident with significant increases in H3K27me3. Biochemical purification of a VRN5 complex showed that during prolonged cold a PHD-PRC2 complex forms composed of core PRC2 components (VRN2, SWINGER [an E(Z) HMTase homologue], FIE [an ESC homologue], MSI1 [p55 homologue]), and three related PHD finger proteins, VRN5, VIN3, and VEL1. The PHD-PRC2 activity increases H3K27me3 throughout the locus to levels sufficient for stable silencing. Arabidopsis PHD-PRC2 thus seems to act similarly to Pcl-PRC2 of Drosophila and PHF1-PRC2 of mammals. These data show FLC silencing involves changed composition and dynamic redistribution of Polycomb complexes at different stages of the vernalization process, a mechanism with greater parallels to Polycomb silencing of certain mammalian loci than the classic Drosophila Polycomb targets.     

Plant adaptation to fluctuating environment and biomass production are strongly dependent on guard cell potassium channels

At least four genes encoding plasma membrane inward K+ channels (K(in) channels) are expressed in Arabidopsis guard cells. A double mutant plant was engineered by disruption of a major K(in) channel gene and expression of a dominant negative channel construct. Using the patch-clamp technique revealed that this mutant was totally deprived of guard cell K(in) channel (GCK(in)) activity, providing a model to investigate the roles of this activity in the plant. GCK(in) activity was found to be an essential effector of stomatal opening triggered by membrane hyperpolarization and thereby of blue light-induced stomatal opening at dawn. It improved stomatal reactivity to external or internal signals (light, CO2 availability, and evaporative demand). It protected stomatal function against detrimental effects of Na+ when plants were grown in the presence of physiological concentrations of this cation, probably by enabling guard cells to selectively and rapidly take up K+ instead of Na+ during stomatal opening, thereby preventing deleterious effects of Na+ on stomatal closure. It was also shown to be a key component of the mechanisms that underlie the circadian rhythm of stomatal opening, which is known to gate stomatal responses to extracellular and intracellular signals. Finally, in a meteorological scenario with higher light intensity during the first hours of the photophase, GCK(in) activity was found to allow a strong increase (35%) in plant biomass production. Thus, a large diversity of approaches indicates that GCK(in) activity plays pleiotropic roles that crucially contribute to plant adaptation to fluctuating and stressing natural environments.     

Splicing inhibition of U2AF65 leads to alternative exon skipping

U2 snRNP auxiliary factor 65 kDa (U2AF⁶⁵) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF⁶⁵ stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5′ splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF⁶⁵. U2AF⁶⁵ overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF⁶⁵ inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF⁶⁵ effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF⁶⁵ even inhibits general splicing of adenovirus major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that U2AF⁶⁵ possesses a splicing Inhibitory function that leads to alternative exon skipping.Pre-mRNA splicing is a process in which noncoding intron sequences are removed and exon sequences are then ligated together (1, 2). Pre-mRNA splicing is carried out by spliceosome, a large RNA–protein complex that contains five small nuclear ribonucleoproteins (U snRNPs) and more than 100 additional proteins (3). Pre-mRNA splicing occurs in the consensus sequences at the 5′ splice-site, 3′ splice-site, and branch point that are necessary for splicing. The sequence between 3′ AG dinucleotide and branch point is the polypyrimidine (Py) tract that directs spliceosome assembly on the 3′ splice-site. Alternative splicing provides an important regulatory mechanism in higher eukaryotes for multiple proteins produced from a single gene (4, 5).The U2 snRNP auxiliary factor 65 kDa (U2AF⁶⁵) exists as a heterodimer with U2AF³⁵ (6). U2AF⁶⁵ contains three C-terminal RNA recognition motifs (RRMs) and an N-terminal arginine/serine-rich (RS) domain (7, 8). Using U2AF⁶⁵ depletion/adding back technology with in vitro HeLa nuclear extract, it was demonstrated that U2AF⁶⁵ is an essential splicing factor (9). Whereas U2AF⁶⁵ binds to Py tract to promote prespliceosome assembly and branchpoint/U2 snRNA base pairing, U2AF³⁵ plays a role in the 3′ splice-site (10, 11). As U2AF⁶⁵ prefers high C/U-rich sequences in the Py tract, a stronger interaction between U2AF⁶⁵ and Py tract promotes prespliceosome assembly (12). U2AF⁶⁵ is also essential in vertebrate development (13, 14). Its expression level is related to myotonic dystrophy, cystic fibrosis, and cancers (15, 16).Proximal spinal muscular atrophy (SMA) is an autosomal recessive genetic disease (17) and a leading cause of infant mortality. The motor neurons in the anterior horn of spinal cord are severely damaged in patients with type 1 SMA, usually leading to death before age 2 y as a result of a lack of respiratory support (18, 19). In patients with SMA, the SMN1 gene is deleted or mutated, whereas the SMN2 gene, a duplicate of the SMN1 gene, is included (20). SMN2 genomic DNA contains a few nucleotide mutations compared with SMN1 (21, 22). Full-length SMN protein functions in the U snRNP assembly/disassembly, as well as in the β-actin mRNA transport in neurons (23, 24). However, the mutations in SMN2 pre-mRNA cause predominantly skipping of exon 7, which produces SMNΔ7, a truncated and less stable protein with reduced self-oligomerization activity. Alternative exon 7 splicing of SMN pre-mRNA was modulated by orchestrated RNA–protein and protein–protein interactions, secondary structures of RNA, and RNA sequences (25–27). Among the mutations on SMN2 pre-mRNA, the most functionally understood one is the C-to-U point mutation on exon 7, which plays an important role in alternative splicing of exon 7 (25–27). In vitro analysis using HeLa nuclear extract and S100 extract demonstrates that SRSF1 promotes exon 7 inclusion through contacting the enhancer sequence on exon 7 of SMN1 pre-mRNA, and that C-to-U mutation on SMN2 pre-mRNA disrupts SRSF1 binding and then consequently disrupts the enhancer function of SRSF1 (28). However, cell-based analysis shows a different result, indicating that SRSF1 does not play an essential role in SMN exon 7 splicing (29). In contrast, cell transfection analysis demonstrates that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 interacts with the C-to-U mutation on SMN2 pre-mRNA to inhibit exon 7 splicing (29). A possible explanation for these different results is that different analysis systems could provide different conclusions.Although the roles of U2AF⁶⁵ in alternative splicing are verified to some extent, the function and mechanism are unclear. The previous reports have shown that U2AF⁶⁵ roles in alternative splicing are the target of alternative splicing regulatory factors, as demonstrated with increased U2AF⁶⁵ binding by other splicing regulatory proteins (30, 31). More recently, genome-wide analysis has demonstrated that upstream intronic binding of U2AF⁶⁵ interferes with the immediate downstream 3′ splice-site of alternative or constitutive exons to cause exon skipping or inclusion (32). In the SMN pre-mRNA, it was demonstrated that U2AF⁶⁵ interacts more strongly with the SMN1 Py tract than the SMN2 Py tract (33). However, it is unclear how U2AF⁶⁵ itself regulates alternative splicing.Here we identified the function of U2AF⁶⁵ in the alternative splicing. Through siRNA-knockdown and overexpression of U2AF⁶⁵, we show that U2AF⁶⁵ promotes alternative exon exclusion of both SMN2 and SMN1 pre-mRNA. Mutations of 5′ splice-site in exon 7 to a higher score sequence abolished the U2AF⁶⁵ effects. Highly expressed U2AF⁶⁵ also represses splicing of exon 7, flanking introns in three or two exon contexts. Strikingly, U2AF⁶⁵ also inhibits intron splicing of adenovirus major late (Ad ML) and β-globin pre-mRNA. In addition, U2AF⁶⁵ selectively increases its own binding on the weaker Py tract sequence, but not the stronger Py tract. Our results support the conclusion that the U2AF⁶⁵ activity in promoting alternative exon skipping comes from its own splicing inhibitory activity.