Histone mRNAs are rapidly degraded at the end of S phase or when DNA replication is inhibited. Histone mRNAs end in a conserved stem-loop rather than a poly(A) tail. Degradation of histone mRNAs requires the stem-loop sequence, which binds the stem-loop-binding protein (SLBP), active translation of the histone mRNA, and the location of the stem-loop close to the termination codon. We report that the initial step in histone mRNA degradation is the addition of uridines to the 3 end of the histone mRNA, both after inhibition of DNA replication and at the end of S phase. Lsm1 is required for histone mRNA degradation and is present in a complex containing SLBP on the 3 end of histone mRNA after inhibition of DNA replication. We cloned degradation intermediates that had been partially degraded from both the 5 and the 3 ends. RNAi experiments demonstrate that both the exosome and 5-to-3 decay pathway components are required for degradation, and individual histone mRNAs are then degraded simultaneously 5 to 3 and 3 to 5.[Keywords: Histone; cell cycle; mRNA stability; decapping; exosome; oligouridylation] Supplemental material is available at http://www.genesdev.org.

The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. We have found that the human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein.The circadian and cell cycles are two global regulatory systems that have pervasive effects on organismal and cellular physiology. Circadian rhythm is the oscillation in the physiology and behavior of organisms with a 24-h periodicity (17,33,40). The rhythm consists of light and dark phases which coincide with the phases of the solar day. Cell cycle checkpoints are regulatory pathways that ensure completion of biochemical reactions unique to each phase of the cell cycle (G 1 , S, G 2 , and M in proliferating mammalian cells) prior to initiation of subsequent phases (26,30,35,41). While these two regulatory systems involve distinct mechanisms, there is some evidence that these cycles are linked. Most mammalian diploid cells exhibit an approximately 24-h cell cycle period, and the circadian clock has been implicated in regulation of the phases of cell division (3). The emerging field of chronotherapy aims to coordinate the time of delivery of chemotherapeutic drugs with the circadian and cell cycles so as to minimize side effects while optimizing therapeutic efficacy (4).Although a few recent studies have shown that some cell proliferation and cell cycle checkpoint genes in mammals (such as c-myc, Wee1, and cyclin D1) are first-and second-order clock-controlled genes (10, 23), the circadian cycle-cell cycle connection remains ill-defined. Here we present evidence that the mammalian Timeless (Tim) protein (18, 36), which appears to be required for a robust circadian rhythm (1), is also a core component of the cell cycle checkpoint system, suggesting a possibly more intimate and direct connection between the circadian cycle and cell cycle checkpoints in mammals.Despite its initial identification as a homologue of the Drosophila clock protein Tim, the closest phylogenetic relatives of the mammalian Tim protein are actually cell cycle-related proteins: budding yeast Tof1 (9, 32), fission yeast Swi1 (29,19), Caenorhabditis elegans TIM-1 (5), and Drosophila Tim-2/Timeout (dTim2/dTimeout) (2). Tof1 and Swi1 have been implicated in DNA damage checkpoint activation as mediators, and Swi1 plays an additional role in preventing replication fork collapse (29). TIM-1 is essential for chromosome cohesion in C. elegans, and Timeless null mutation results in embryonic lethality in both C. elegans (5) and mice (12). Based on these find...

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared to their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4+ NPCs with a pharmacological inhibitor of glycogen synthase kinase 3 (GSK3), a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD-patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

Histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated. Synthesis of mature histone mRNA requires only a single processing reaction: an endonucleolytic cleavage between a conserved stem-loop and a purine-rich downstream element to form the 39 end. The stem-loop binding protein (SLBP) is required for processing, and following processing, histone mRNA is transported to the cytoplasm, where SLBP participates in translation of the histone mRNA and is also involved in regulation of histone mRNA degradation. Here we present an analysis of histone mRNA metabolism in cells with highly reduced levels of SLBP using RNA interference. Knocking down SLBP in U2OS cells results in a reduction in the rate of cell growth and an accumulation of cells in S-phase. Surprisingly, there is only a modest (twofold) decrease in histone mRNA levels. Much of histone mRNA in the SLBP knockdown cells is properly processed but is retained in the nucleus. The processed histone mRNA in SLBP knockdown cells is not rapidly degraded when DNA replication is inhibited. These results suggest a previously undescribed role for SLBP in histone mRNA export.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

Our study suggests that CLN5 mutations 1) are more common in patients with neuronal ceroid lipofuscinosis (NCL) than previously reported, 2) are found in non-Finnish NCL patients of broad ethnic diversity, and 3) can be identified in NCL patients with disease onset in adult and juvenile epochs. CLN5 genetic testing is warranted in a wider population with clinical and pathologic features suggestive of an NCL disorder.

Norrie disease (ND) is an X-linked recessive disorder characterized by congenital blindness, progressive sensorineural hearing loss and cognitive impairment. The ocular phenotype has been well described, while the extraocular manifestations of the disorder are not well understood. We present the data from the Norrie Disease Registry, which consists of 56 patients with detailed clinical histories and genotype data. This study represents the largest, detailed investigation into the phenotypic spectrum of ND to date and more importantly expands knowledge of the extraocular clinical manifestations. We identify several novel aspects of the syndrome that will improve the management of these patients. In particular, we expand our understanding of the neurologic manifestations in ND and identify a chronic seizure disorder in approximately 10% of all patients. In addition, details of the hearing phenotype are described including the median age of onset (12 years of age) and how genotype affects onset. Moreover, we find vascular disease to be a significant component of ND; and vascular health should be, in the future, a component of patient clinical care. In summary, the results expand our understanding of the phenotypic variability and genotypic heterogeneity in ND patients.

porting the assertion that this parasitic paramoeba may be the primary cause of the 1999 lobster mass mortality.