DNA looping-mediated repression by histone-like protein H-NS: specific requirement of Esigma70 as a cofactor for looping.

Shin, Minsang,Song, Miryoung,Rhee, Joon Haeng,Hong, Yeongjin,Kim, You-Jin,Seok, Yeong-Jae,Ha, Kwon-Soo,Jung, Se-Hui,Choy, Hyon E Cold Spring Harbor Laboratory in association with 2005 Genes & development Vol.19 No.19

<P>Transcription initiation by RNA polymerase (RNP) carrying the house-keeping sigma subunit, sigma70 (Esigma70), is repressed by H-NS at a number of promoters including hdeABp in Escherichia coli, while initiation with RNP carrying the stationary phase sigma, sigma38 (Esigma38), is not. We investigated the molecular mechanism of selective repression by H-NS to identify the differences in transcription initiation by the two forms of RNPs, which show indistinguishable promoter selectivities in vitro. Using hdeABp as a model promoter, we observed with purified components that H-NS, acting at a sequence centered at -118, selectively repressed transcription by Esigma70. This selective repression is attributed to the differences in the interactions between hdeABp and the two forms of RNPs, since no other factor is required for the repression. We observed that the two forms of RNPs could form an open initiation complex (RP(O)) at hdeABp, but that Esigma70 failed to initiate transcription in the presence of H-NS. Interestingly, KMnO4 assays and high-resolution atomic force microscopy (AFM) revealed that hdeABp DNA wrapped around Esigma70 more tightly than around Esigma38, resulting in the potential crossing over of the DNA arms that project out of Esigma70 . RP(O) but not out of Esigma38 . RP(O). Based on these observations, we postulated that H-NS bound at -118 laterally extends by the cooperative recruitment of H-NS molecules to the promoter-downstream sequence joined by wrapping of the DNA around Esigma70 . RP(O), resulting in effective sealing of the DNA loop and trapping of Esigma70. Such a ternary complex of H-NS . Esigma70 hdeABp was demonstrated by AFM. In this case, therefore, Esigma70 acts as a cofactor for DNA looping. Expression of this class of genes by Esigma38 in the stationary phase is not due to its promoter specificity but to the architecture of the promoter . Esigma38 complex.</P>

Retinal degeneration triggered by inactivation of PTEN in the retinal pigment epithelium.

Kim, Jin Woo,Kang, Kyung Hwa,Burrola, Patrick,Mak, Tak W,Lemke, Greg Cold Spring Harbor Laboratory in association with 2008 Genes & development Vol.22 No.22

<P>Adhesion between epithelial cells mediates apical-basal polarization, cell proliferation, and survival, and defects in adhesion junctions are associated with abnormalities from degeneration to cancer. We found that the maintenance of specialized adhesions between cells of the retinal pigment epithelium (RPE) requires the phosphatase PTEN. RPE-specific deletion of the mouse pten gene results in RPE cells that fail to maintain basolateral adhesions, undergo an epithelial-to-mesenchymal transition (EMT), and subsequently migrate out of the retina entirely. These events in turn lead to the progressive death of photoreceptors. The C-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding domain of PTEN is essential for the maintenance of RPE cell junctional integrity. Inactivation of PTEN, and loss of its interaction with junctional proteins, are also evident in RPE cells isolated from ccr2(-/-) mice and from mice subjected to oxidative damage, both of which display age-related macular degeneration (AMD). Together, these results highlight an essential role for PTEN in normal RPE cell function and in the response of these cells to oxidative stress.</P>

Small RNAs just got bigger: Piwi-interacting RNAs (piRNAs) in mammalian testes.

Cold Spring Harbor Laboratory in association with 2006 Genes & development Vol.20 No.15

<P>Small RNAs constitute a large family of regulatory molecules with diverse functions in eukaryotes. Hallmarks of small RNAs are their dependence on double-stranded RNAs (dsRNA)-specific RNase III-type enzymes for biogenesis and their association with Argonaute family proteins for the silencing process. At least two classes of small RNAs have previously been described: microRNAs (miRNAs) derived from hairpin-shaped precursors and small interfering RNAs (siRNAs) generated from long dsRNAs. Recent articles reported a novel class of small RNAs that are expressed specifically and abundantly in the spermatogenic cells of mice. These RNAs are bigger (26-31 nucleotides [nt]) than most previously described small RNAs (21-23 nt) and are associated with Piwi-subclade members of the Argonaute protein family. Although the biogenesis and function of these RNAs are yet to be determined, these findings may add new dimensions in small RNA biology and germline cell biology.</P>

Rhythmic control of AANAT translation by hnRNP Q in circadian melatonin production.

Kim, Tae-Don,Woo, Kyung-Chul,Cho, Sungchan,Ha, Dae-Cheong,Jang, Sung Key,Kim, Kyong-Tai Cold Spring Harbor Laboratory in association with 2007 Genes & development Vol.21 No.7

<P>The circadian rhythm of pineal melatonin requires the nocturnal increment of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) protein. To date, only limited information is available in the critical issue of how AANAT protein expression is up-regulated exclusively at night regardless of its species-specific mRNA profiles. Here we show that the circadian timing of AANAT protein expression is regulated by rhythmic translation of AANAT mRNA. This rhythmic control is mediated by both a highly conserved IRES (internal ribosome entry site) element within the AANAT 5' untranslated region and its partner hnRNP Q (heterogeneous nuclear ribonucleoprotein Q) with a peak in the middle of the night. Consistent with the enhancing role of hnRNP Q in AANAT IRES activities, knockdown of the hnRNP Q level elicited a dramatic decrease of peak amplitude in the AANAT protein profile parallel to reduced melatonin production in pinealocytes. This translational regulation of AANAT mRNA provides a novel aspect for achieving the circadian rhythmicity of vertebrate melatonin.</P>

The U2AF35-related protein Urp contacts the 3' splice site to promote U12-type intron splicing and the second step of U2-type intron splicing.

Shen, Haihong,Zheng, Xuexiu,Luecke, Stephan,Green, Michael R Cold Spring Harbor Laboratory in association with 2010 Genes & development Vol.24 No.21

<P>The U2AF35-related protein Urp has been implicated previously in splicing of the major class of U2-type introns. Here we show that Urp is also required for splicing of the minor class of U12-type introns. Urp is recruited in an ATP-dependent fashion to the U12-type intron 3' splice site, where it promotes formation of spliceosomal complexes. Remarkably, Urp also contacts the 3' splice site of a U2-type intron, but in this case is specifically required for the second step of splicing. Thus, through recognition of a common splicing element, Urp facilitates distinct steps of U2- and U12-type intron splicing.</P>

A new MIF4G domain-containing protein, CTIF, directs nuclear cap-binding protein CBP80/20-dependent translation.

Kim, Kyoung Mi,Cho, Hana,Choi, Kobong,Kim, Jaedong,Kim, Bong-Woo,Ko, Young-Gyu,Jang, Sung Key,Kim, Yoon Ki Cold Spring Harbor Laboratory in association with 2009 Genes & development Vol.23 No.17

<P>During or right after mRNA export via the nuclear pore complex (NPC) in mammalian cells, mRNAs undergo translation mediated by nuclear cap-binding proteins 80 and 20 (CBP80/20). After CBP80/20-dependent translation, CBP80/20 is replaced by cytoplasmic cap-binding protein eIF4E, which directs steady-state translation. Nonsense-mediated mRNA decay (NMD), one of the best-characterized mRNA surveillance mechanisms, has been shown to occur on CBP80/20-bound mRNAs. However, despite the tight link between CBP80/20-dependent translation and NMD, the underlying molecular mechanism and cellular factors that mediate CBP80/20-dependent translation remain obscure. Here, we identify a new MIF4G domain-containing protein, CTIF (CBP80/20-dependent translation initiation factor). CTIF interacts directly with CBP80 and is part of the CBP80/20-dependent translation initiation complex. Depletion of endogenous CTIF from an in vitro translation system selectively blocks the translation of CBP80-bound mRNAs, while addition of purified CTIF restores it. Accordingly, down-regulation of endogenous CTIF abrogates NMD. Confocal microscopy shows that CTIF is localized to the perinuclear region. Our observations demonstrate the existence of CBP80/20-dependent translation and support the idea that CBP80/20-dependent translation is mechanistically different from steady-state translation through identification of a specific cellular protein, CTIF.</P>

Bacterial-modulated host immunity and stem cell activation for gut homeostasis.

Cold Spring Harbor Laboratory in association with 2009 Genes & development Vol.23 No.19

<P>Although it is widely accepted that dynamic cross-talk between gut epithelia and microorganisms must occur to achieve gut homeostasis, the critical mechanisms by which gut-microbe interactions are regulated remain uncertain. In this issue of Genes & Development, Buchon and colleagues (pp. 2333-2344) revealed that the reaction of the gut to microorganisms is not restricted to activating immune systems, but extends to integrated responses essential for gut tissue homeostasis, including self-renewal and the differentiation of stem cells. Further investigation of the connection between immune response and stem cell regulation at the molecular level in the microbe-laden mucosal epithelia will accelerate our understanding of the regulatory mechanisms of gut homeostasis and of the pathogenesis of diseases such as chronic inflammatory diseases and colorectal cancers.</P>

The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome.

Song, Eun Joo,Werner, Shannon L,Neubauer, Jakob,Stegmeier, Frank,Aspden, Julie,Rio, Donald,Harper, J Wade,Elledge, Stephen J,Kirschner, Marc W,Rape, Michael Cold Spring Harbor Laboratory in association with 2010 Genes & development Vol.24 No.13

<P>The spliceosome, a dynamic assembly of proteins and RNAs, catalyzes the excision of intron sequences from nascent mRNAs. Recent work has suggested that the activity and composition of the spliceosome are regulated by ubiquitination, but the underlying mechanisms have not been elucidated. Here, we report that the spliceosomal Prp19 complex modifies Prp3, a component of the U4 snRNP, with nonproteolytic K63-linked ubiquitin chains. The K63-linked chains increase the affinity of Prp3 for the U5 snRNP component Prp8, thereby allowing for the stabilization of the U4/U6.U5 snRNP. Prp3 is deubiquitinated by Usp4 and its substrate targeting factor, the U4/U6 recycling protein Sart3, which likely facilitates ejection of U4 proteins from the spliceosome during maturation of its active site. Loss of Usp4 in cells interferes with the accumulation of correctly spliced mRNAs, including those for alpha-tubulin and Bub1, and impairs cell cycle progression. We propose that the reversible ubiquitination of spliceosomal proteins, such as Prp3, guides rearrangements in the composition of the spliceosome at distinct steps of the splicing reaction.</P>

Crystal structure of the human GINS complex.

Choi, Jung Min,Lim, Hye Seong,Kim, Jeong Joo,Song, Ok-Kyu,Cho, Yunje Cold Spring Harbor Laboratory in association with 2007 Genes & development Vol.21 No.11

<P>The GINS complex mediates the assembly of the MCM2-7 (minichromosome maintenance) complex with proteins in a replisome progression complex. The eukaryotic GINS complex is composed of Sld5, Psf1, Psf2, and Psf3, which must be assembled for cell proliferation. We determined the crystal structure of the human GINS complex: GINS forms an elliptical shape with a small central channel. The structures of Sld5 and Psf2 resemble those of Psf1 and Psf3, respectively. In addition, the N-terminal and C-terminal domains of Sld5/Psf1 are permuted in Psf2/Psf3, which suggests that the four proteins have evolved from a common ancestor. Using a structure-based mutational analysis, we identified the functionally critical surface regions of the GINS complex.</P>

A novel cell-to-cell trafficking assay indicates that the KNOX homeodomain is necessary and sufficient for intercellular protein and mRNA trafficking.

Kim, Jae-Yean,Rim, Yeonggil,Wang, Jing,Jackson, David Cold Spring Harbor Laboratory in association with 2005 Genes & development Vol.19 No.7

<P>Cell-to-cell trafficking of regulatory proteins is a novel mechanism for communication during cell fate specification in plants. Although several developmental proteins traffic cell-to-cell, no signals that are both necessary and sufficient for this function in developmental proteins have been described. We developed a novel trafficking assay using trichome rescue in Arabidopsis. Fusion to KNOTTED1 (KN1) conferred gain-of-trafficking function to the cell-autonomous GLABROUS1 (GL1) protein. We show that the KNOX homeodomain (HD) is necessary and sufficient for intercellular trafficking, identifying a novel function for the HD as the minimal sequence required for trafficking of KN1 and its associated mRNA.</P>