PURPOSE. BCL-X L , an anti-apoptotic member of the BCL-2 family proteins and a cell death/survival checkpoint regulator, was shown to be upregulated in bright light-stressed mouse photoreceptors during an investigation of bright light-induced protein expression. To investigate the significance of BCL-X L upregulation in the bright light damage model, the Bcl-x gene was disrupted specifically in mouse rod photoreceptors, and the effect of Bcl-x disruption was characterized on retinal apoptosis, function, and morphology. METHODS. Rod-specific Bcl-x knockout mice, generated by mating mouse opsin promoter-controlled Cre mice with floxed Bcl-x mice, were subjected to bright light stress. Retinal apoptosis in the bright light-stressed conditional Bcl-x knockout mice was characterized with TUNEL, DNA fragmentation, and nuclear staining assays. Photoreceptor structural and functional integrity in the bright light-stressed conditional Bcl-x knockout mice was determined by measuring photoreceptor outer nuclear layer (ONL) thickness and electroretinography amplitudes. RESULTS. Disruption of Bcl-x in rod photoreceptors caused increased photoreceptor apoptosis, decreased retinal function, and decreased ONL thickness in bright light-stressed mice. CONCLUSIONS. The loss of BCL-X L increased rod photoreceptor susceptibility to bright light stress. Although the biochemical mechanism(s) of BCL-X L in photoreceptor death or survival has not been investigated extensively, results of the present study suggest that BCL-X L , a cell survival/death checkpoint regulator, is involved in photoreceptor survival under bright light stress.

PURPOSE. BCL-X L , an anti-apoptotic member of the BCL-2 family proteins and a cell death/survival checkpoint regulator, was shown to be upregulated in bright light-stressed mouse photoreceptors during an investigation of bright light-induced protein expression. To investigate the significance of BCL-X L upregulation in the bright light damage model, the Bcl-x gene was disrupted specifically in mouse rod photoreceptors, and the effect of Bcl-x disruption was characterized on retinal apoptosis, function, and morphology. METHODS. Rod-specific Bcl-x knockout mice, generated by mating mouse opsin promoter-controlled Cre mice with floxed Bcl-x mice, were subjected to bright light stress. Retinal apoptosis in the bright light-stressed conditional Bcl-x knockout mice was characterized with TUNEL, DNA fragmentation, and nuclear staining assays. Photoreceptor structural and functional integrity in the bright light-stressed conditional Bcl-x knockout mice was determined by measuring photoreceptor outer nuclear layer (ONL) thickness and electroretinography amplitudes. RESULTS. Disruption of Bcl-x in rod photoreceptors caused increased photoreceptor apoptosis, decreased retinal function, and decreased ONL thickness in bright light-stressed mice. CONCLUSIONS. The loss of BCL-X L increased rod photoreceptor susceptibility to bright light stress. Although the biochemical mechanism(s) of BCL-X L in photoreceptor death or survival has not been investigated extensively, results of the present study suggest that BCL-X L , a cell survival/death checkpoint regulator, is involved in photoreceptor survival under bright light stress.

Genetic reference populations in model organisms are critical resources for systems genetic analysis of disease related phenotypes. The breeding history of these inbred panels may influence detectable allelic and phenotypic diversity. The existing panel of common inbred strains reflects historical selection biases, and existing recombinant inbred panels have low allelic diversity. All such populations may be subject to consequences of inbreeding depression. The Collaborative Cross (CC) is a mouse reference population with high allelic diversity that is being constructed using a randomized breeding design that systematically outcrosses eight founder strains, followed by inbreeding to obtain new recombinant inbred strains. Five of the eight founders are common laboratory strains, and three are wild-derived. Since its inception, the partially inbred CC has been characterized for physiological, morphological, and behavioral traits. The construction of this population provided a unique opportunity to observe phenotypic variation as new allelic combinations arose through intercrossing and inbreeding to create new stable genetic combinations. Processes including inbreeding depression and its impact on allelic and phenotypic diversity were assessed. Phenotypic variation in the CC breeding population exceeds that of existing mouse genetic reference populations due to both high founder genetic diversity and novel epistatic combinations. However, some focal evidence of allele purging was detected including a suggestive QTL for litter size in a location of changing allele frequency. Despite these inescapable pressures, high diversity and precision for genetic mapping remain. These results demonstrate the potential of the CC population once completed and highlight implications for development of related populations.

The onset of the daily wheel running bout precedes dark onset by several hours in the early runner genetic variant of mice. Here, we test the hypothesis that timed daily administration of a melatonin agonist, ramelteon, or a benzodiazepine, triazolam, normalizes the timing of daily wheel-running rhythms in early runner mice. The daily profiles of wheel-running activity of early runner mice were monitored continuously in a 12:12 light/dark cycle. Wheel running was assessed before, during and after timed daily oral administration of saline vehicle (n = 12), ramelteon (10 mg/kg, n = 12), or triazolam (1 mg/kg, n = 12). The timing of wheel-running rhythms relative to the light/dark cycle was used as a measure of the timing of wake onset. Under baseline conditions, early runner mice entrained to a light/dark cycle at an advanced phase, approximately 3 h before dark onset, on average. Triazolam, but not ramelteon, suppressed wheel-running acutely when administered just prior to the time at which wheel-running onset had occurred under baseline conditions. On a washout day under a light/dark cycle subsequent to one week of once daily administration, the onset of wheel-running was delayed relative to baseline in both ramelteon-treated mice and triazolam-treated mice. In constant dark subsequent to a second week of once daily administration, the onset of wheel-running activity was not affected by either compound.

The adaptive value of the endogenous circadian clock arises from its ability to synchronize (i.e., entrain) to external light-dark (LD) cycles at an appropriate phase. Studies have suggested that advanced circadian phase alignment might result from shortening of the period length of the clock. Here we explore mechanisms that contribute to an early activity phase in CAST/EiJ (CAST) mice. Methods: We investigated circadian rhythms of wheel-running activity in C57BL/6J (B6), CAST and 2 strains of B6.CAST congenic mice, which carry CAST segments introgressed in a B6 genome. Results: When entrained, all CAST mice initiate daily activity several hours earlier than normal mice. This difference could not be explained by alterations in the endogenous period, as activity onset did not correlate with period length. However, the photic phase-shifting responses in these mice were phase-lagged by 3 hours relative to their activity. Attenuated light masking responses were also found in CAST mice, which allow for activity normally inhibited by light. A previously identified quantitative trait locus (QTL), Era1, which contributes to the early activity trait, was confirmed and refined here using two B6.CAST congenic strains. Surprisingly, these B6.CAST mice exhibited longer rather than shorter endogenous periods, further demonstrating that the advanced phase in these mice is not due to alterations in period. Conclusions: CAST mice have an advanced activity phase similar to human advanced sleep phase syndrome. This advanced phase is not due to its shorter period length or smaller light-induced phase shifts, but appears to be related to both light masking and altered coupling of the circadian pacemaker with various outputs. Lastly, a QTL influencing this trait was confirmed and narrowed using congenic mice as a first step toward gene identification. Citation: Jiang P; Striz M; Wisor JP; O'Hara BF. Behavioral and genetic dissection of a mouse model for advanced sleep phase syndrome. SLEEP 2011;34(1):39-48.

Profound disruptions of circadian rhythms and sleep/wake cycles constitute a major cause of institutionalization of AD patients. This study investigated whether a rodent model of AD, APP NLH/NLH /PS-1 P264L/264L (APPxPS1) mice, exhibits circadian alterations. The APPxPS1 mice were generated using CD-1/129 mice and Cre-lox knock-in technology to "humanize" the mouse amyloid (A)β sequence and create a presenilin-1 mutation identified in familial early-onset AD patients. APPxPS1 and WT mice of several ages (~4, 11, and 15 months) were monitored for circadian rhythms in wheel running, cage activity, and sleep:wake behavior. After rhythm assessment, the mice were euthanized at zeitgeber time (ZT) 2 or 10 (i.e., 2 or 10 h after lights-on) and brains were dissected. Amyloidβ levels were measured in cortical samples and brain sections of the hypothalamus and hippocampus were prepared and used for in situ hybridization of circadian or neuropeptide genes. The most significant effects of the APPxPS1 transgenes were phase delays of~2 h in the onset of daytime wakefulness bouts (Pb 0.005) and peak wakefulness (Pb 0.02), potentially relevant to phase delays previously reported in AD patients. However, genotype did not affect the major activity peaks or phases of wheel running, wake, or general movement, which were bimodal with dominant dawn and dusk activity. Expression of Period 2 in the suprachiasmatic nucleus was affected by ZT (Pb 0.0001) with a marginal interaction effect of age, genotype, and ZT (Pb 0.08). A separate analysis of the old animals indicated a robust interaction between ZT and genotype, as well as main effects of these parameters. Aging also altered sleep (e.g., bout length and amount of daytime sleep) and the amount of wheel running and cage activity. In conclusion, the APPxPS1 knock-in mice exhibit some alterations in their sleep:wake rhythm and clock gene expression, but do not show robust, genotype-related changes in activity rhythms. The prominent daytime activity peaks shown by the background strain complicate the use of these APPxPS1 knock-in mice for investigations of circadian activity rhythms in AD. In addition to this unusual activity pattern, lack of hyperactivity differentiates the APPxPS1 knock-in mice from other transgenic AD models.

Parallel investigations of yeast and metazoan pre-mRNA splicing have documented enormous complexity in the nucleic acid and protein components of the cellular splicing apparatus, the spliceosome. The degree to which yeast and metazoan spliceosomal proteins differ in composition and structure is currently unknown. In this report we demonstrate that the human small nuclear ribonucleoprotein (snRNP) polypeptide Dl complements the cell lethality, splicing deficiency, and snRNA instability phenotypes associated with a yeast smdl null allele. Mutational analysis of yeast SMD1, guided by a comparison of the predicted yeast and human proteins, reveals that a large, nonconserved portion of Smdl p is dispensable for biological activity. These observations firmly establish Dl as an essential component of the cellular splicing apparatus and suggest that yeast and metazoa are remarkably similar in the polypeptides guiding early snRNP assembly.

Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 59 splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition.

The U1 snRNP functions to nucleate spliceosome assembly on newly transcribed pre-mRNA. Saccharomyces cerevisiae is unusual among eukaryotes in the greatly extended length of its U1 snRNA and the apparent increased polypeptide complexity of the corresponding U1 snRNP. In this paper, we report the identification of a novel U1 snRNP protein, Prp42p, with unexpected properties. Prp42p was identified by its surprising structural similarity to the essential U1 snRNP protein, Prp39p. Both Prp39p and Prp42p possess multiple copies of a variant tetratricopeptide repeat, an element implicated in a wide range of protein assembly events. Yeast strains depleted of Prp42p by transcriptional repression of a GAL1::PRP42 fusion gene arrest for splicing prior to pre-mRNA 5 splice site cleavage. Prp42p was not observed in a recent biochemical analysis of purified U1 snRNPs from S. cerevisiae (28). Nevertheless, antibodies directed against an epitope-tagged version of Prp42p specifically precipitate U1 snRNA from yeast extracts. Furthermore, Prp42p is required for U1 snRNP biogenesis, because yeast strains depleted of Prp42p formed incomplete U1 snRNPs that failed to produce stable complexes with pre-mRNA in vitro. The evidence shows that Prp39p and Prp42p are both required to configure the atypical yeast U1 snRNP into a structure compatible with its evolutionarily conserved role in pre-mRNA splicing.

A chemical modification/interference assay was used to determine the yeast pre-mRNA sequence requirements for in vitro spliceosome assembly and splicing. Modifications of any of the nucleotides within the 5' splice site and branch point (TACTAAC box) consensus sequences as well as less conserved intron and exon positions were found to inhibit assembly and/or splicing. The interference pattern of the 5' splice site and TACTAAC box lesions increased as spliceosome assembly proceeded (complex III-> complex I-* complex II) and as splicing proceeded, suggesting that these sequence elements play multiple roles in the assembly of yeast spliceosomes and in the removal of intervening sequences. Furthermore, modification (or mutation) of a TACTAAC-Iike sequence upstream of the branch point was found to inhibit the rate of spliceosome assembly, implying a possible role for degenerate branch point sequences in modulating the efficiency of spliceosome assembly.

To address the mechanisms that underlie splice site selection and splice site partner assignment, we analyzed the splicing of yeast (Saccharomyces cerevisiae) transcripts containing splice site region duplications. When the 5'-splice site region was duplicated, both sites were utilized to the same extent, indicating little or no influence of proximity on 5'-splice site choice. However, the effect of a 5'-mutant site was greatly enhanced by the presence of an adjacent wild-type site, and this effect was reversed by the restoration of base-pairing with U1 snRNA. 3'-Splice site choice was apparently influenced by proximity, as the site closest to the 5'-splice site was greatly preferred. Studies with strains carrying some U1 snRNA mutations showed an increase in the use of the distal 3'-splice site, indicating a role for U1 snRNP in 3'-splice site selection. The data are compared with those from mammalian splice site choice experiments and suggest mechanisms that influence differential splice site choice as well as exon skipping.

Prp43p catalyzes essential steps in pre-mRNA splicing and rRNA biogenesis. In splicing, Spp382p stimulates the Prp43p helicase to dissociate the postcatalytic spliceosome and, in some way, to maintain the integrity of the spliceosome assembly. Here we present a dosage interference assay to identify Spp382pinteracting factors by screening for genes that when overexpressed specifically inhibit the growth of a conditional lethal prp38-1 spliceosome assembly mutant in the spp382-1 suppressor background. Identified, among others, are genes encoding the established splicing factors Prp8p, Prp9p, Prp11p, Prp39p, and Yhc1p and two poorly characterized proteins with possible links to splicing, Sqs1p and Cwc23p. Sqs1p copurifies with Prp43p and is shown to bind Prp43p and Spp382p in the two-hybrid assay. Overexpression of Sqs1p blocks pre-mRNA splicing and inhibits Prp43p-dependent steps in rRNA processing. Increased Prp43p levels buffer Sqs1p cytotoxicity, providing strong evidence that the Prp43p DExD/H-box protein is a target of Sqs1p. Cwc23p is the only known yeast splicing factor with a DnaJ motif characteristic of Hsp40like chaperones. We show that similar to SPP382, CWC23 activity is critical for efficient pre-mRNA splicing and intron metabolism yet, surprisingly, this activity does not require the canonical DnaJ/Hsp40 motif. These and related data establish the value of this dosage interference assay for finding genes that alter cellular splicing and define Sqs1p and Cwc23p as prospective modulators of Spp382p-stimuated Prp43p function. E IGHT phylogenetically conserved DExD/H-box proteins act at discrete steps to regulate the assembly, activation, and dissociation of the splicing apparatus (reviewed in Brow 2002; Konarska and Query 2005; Linder 2006). How these RNA-dependent ATPases are temporally and functionally regulated remains poorly understood. One putative regulator, the 83-kDa Spp382/Ntr1 protein (henceforth referred to by the Saccharomyces Genome Database standard name, Spp382p), was discovered in a screen for mutants capable of suppressing defects in yeast spliceosome assembly (Pandit et al. 2006). While spp382 null alleles are lethal, partial loss of function suppresses mutations in several other splicing factors, including the genes encoding the essential spliceosomal proteins Prp8p and Prp38p. Spp382p is a spliceosomal protein that binds the Prp43p DExD/H-box protein to promote efficient dissociation of spliceosomal factors after completion of splicing in vitro (Tsai et al. 2005). Some but not all spp382 mutants also accumulate the excised intron product of splicing in vivo, ostensibly due to protection of the intron within a hyperstabilized spliceosome (Pandit et al. 2006; Tanaka et al. 2007). The suppression of spliceosome assembly defects by spp382 mutation is proposed to occur by impairing Spp382p-stimulated dissociation of kinetically impaired or otherwise inefficient spliceosomes by Prp43p (Pandit et al. 2006). Consistent with this hypothesis, prp43 mutations also suppress spliceosome assembly defects in a manner that, within limits, is inversely proportional to the residual ATPase activity of Prp43p (Pandit et al. 2006). In this light, it is possible that the Spp382 and Prp43 proteins are components of the ''discard pathway'' for spliceosome dissociation predicted by the kinetic proofreading model of DExD/H-box protein function (Burgess et al. 1990; Konarska and Query 2005). Recently, several groups made the surprising observation that the Prp43p splicing factor is also required for ribosome biogenesis. Mutations in PRP43 inhibit 35S pre-rRNA cleavage and limit downstream steps in this processing pathway (

The U2 snRNP particle is an essential component of the eukaryotic pre-mRNA splicing apparatus, the spliceosome. Natural and semisynthetic inhibitors that bind the SF3b subunit of the U2 snRNP block splicing and prompt nuclear export of intron-bearing precursors, defining a new mode of action in anticancer drugs.

Core snRNP proteins bind snRNA through the conserved Sm site, PuA(U) n≥3 GPu. While yeast U1 snRNA has three matches to the Sm consensus, the U1 3′-terminal Sm site was found to be both necessary and sufficient for U1 function. Mutation of this site inhibited pre-mRNA splicing, blocked cell division and resulted in the accumulation of two 3′-extended forms of the U1 snRNA. Cells which harbor the Sm site mutation lack mature U1 RNA (U1α) but have a minor polyadenylated species, U1γ, and a prominent, non-polyadenylated species, U1β. Metabolic depletion of the essential Sm core protein, Smd1p, also resulted in the increased accumulation of U1β and U1γ. In vitro, synthetic U1 precursors were cleaved by Rnt1p (RNase III) very near the U1β 3′-end observed in vivo. We propose that U1β is an Rnt1p-cleaved intermediate and that U1 maturation to the U1α form occurs through an Sm-sensitive step. Interestingly, both U1α and a second, much longer RNA, U1ε, were produced in an rnt1 mutant strain. These results suggest that yeast U1 snRNA processing may progress through Rnt1p-dependent and Rnt1pindependent pathways, both of which require a functional Sm site for final snRNA maturation.

Evidence is presented for the existence of a specific intronintron interaction, necessary for the formation of the branched product in the self-splicing reaction of a group II yeast mitochondrial intron. Trans-splicing reactions involving two RNA molecules (5'exon with covalently linked regions of intron and intron with covalently linked 3'exon) show that the presence of portions of intron domain I on the 5' molecule is necessary for the formation of branched products which are not seen with shorter 5' molecules. Modification/interference reactions show regions necessary for branch-formation and support a major role for specific regions of intron domain I. Further experiments, utilizing a truncated 3' molecule that is missing the conserved branchpoint nucleotide, indicate that domain VI may be required for a successful domain I interaction. A model for the formation of a proper branched structure includes implications for both cis and trans configurations.