Toll-like receptor 3 activation is required for normal skin barrier repair following UV damage.

UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3). This release of ncRNA triggers inflammation in the skin following UV damage. Recently, TLR3 activation was also shown to aid wound repair and increase the expression of genes associated with permeability barrier repair. Here, we sought to test whether skin barrier repair after UVB damage is dependent on the activation of TLR3. We observed that multiple ncRNAs induced expression of skin barrier repair genes, that the TLR3 ligand Poly (I:C) also induced expression and function of tight junctions, and that the ncRNA U1 acts in a TLR3-dependent manner to induce expression of skin barrier repair genes. These observations were shown to have functional relevance as Tlr3-/- mice displayed a delay in skin barrier repair following UVB damage. Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.

Negative regulation of Shh levels by Kras and Fgfr2 during hair follicle development.

Activating mutations in the KRAS oncogene are associated with three related human syndromes, which vary in hair and skin phenotypes depending on the involved allele. How variations in RAS signals are interpreted during hair and skin development is unknown. In this study, we investigated the developmental and transcriptional response of skin and hair to changes in RAS activity, using mouse genetic models and microarray analysis. While activation of Kras (Kras(G12D)) in the skin had strong effects on hair growth and hair shape, steady state changes in downstream RAS/MAPK effectors were subtle and detected only by transcriptional responses. To model the transcriptional response of multiple developmental pathways to active RAS, the effects of growth factor stimulation were studied in skin explants. Here FGF acutely suppressed Shh transcription within 90 min but had significantly less effect on Eda, WNT, Notch or BMP pathways. Furthermore, in vivo Fgfr2 loss-of-function in the ectoderm caused derepression of Shh, revealing a role for FGF in Shh regulation in the hair follicle. These studies define both dosage sensitive effects of RAS signaling on hair morphogenesis and reveal acute mechanisms for fine-tuning Shh levels in the hair follicle.

Rational extension of the ribosome biogenesis pathway using network-guided genetics.

Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics-an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies-to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes-most with human orthologs-to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.

Arx1 is a nuclear export receptor for the 60S ribosomal subunit in yeast.

We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

The specialized cytosolic J-protein, Jjj1, functions in 60S ribosomal subunit biogenesis.

J-proteins and Hsp70 chaperones function together in diverse cellular processes. We identified a cytosolic J-protein, Jjj1, of Saccharomyces cerevisiae that is associated with 60S ribosomal particles. Unlike Zuo1, a 60S subunit-associated J-protein that is a component of the chaperone machinery that binds nascent polypeptide chains upon their exit from the ribosome, Jjj1 plays a role in ribosome biogenesis. Cells lacking Jjj1 have phenotypes very similar to those lacking Rei1, a ribosome biogenesis factor associated with pre-60S ribosomal particles in the cytosol. Jjj1 stimulated the ATPase activity of the general cytosolic Hsp70 Ssa, but not Ssb, Zuo1's ribosome-associated Hsp70 partner. Overexpression of Jjj1, which is normally approximately 40-fold less abundant than Zuo1, can partially rescue the phenotypes of cells lacking Zuo1 as well as cells lacking Ssb. Together, these results are consistent with the idea that Jjj1 normally functions with Ssa in a late, cytosolic step of the biogenesis of 60S ribosomal subunits. In addition, because of its ability to bind 60S subunits, we hypothesize that Jjj1, when overexpressed, is able to partially substitute for the Zuo1:Ssb chaperone machinery by recruiting Ssa to the ribosome, facilitating its interaction with nascent polypeptide chains.

Nuclear recycling of the pre-60S ribosomal subunit-associated factor Arx1 depends on Rei1 in Saccharomyces cerevisiae.

Arx1 and Rei1 are found on late pre-60S ribosomal particles containing the export adaptor Nmd3. Arx1 is related to methionine aminopeptidases (MetAPs), and Rei1 is a C2H2 zinc finger protein whose function in ribosome biogenesis has not been previously characterized. Arx1 and Rei1 localized predominately to the nucleus and cytoplasm, respectively, but could be coimmunoprecipitated, suggesting that they are transiently in the same 60S complex. arx1delta mutants showed a modest accumulation of 60S subunits in the nucleus, suggesting that Arx1 enhances 60S export. Deletion of REI1 led to cold sensitivity and redistribution of Arx1 to the cytoplasm, where it remained bound to free 60S subunits. However, deletion of ARX1 or the fusion of enhanced GFP (eGFP) to Rpl25 suppressed the cold sensitivity of an rei1delta mutant. The presence of eGFP on Rpl25 or its neighboring protein Rpl35 reduced the binding of Arx1 to 60S subunits, suggesting that Arx1 binds to 60S subunits in the vicinity of the exit tunnel. Mutations in Arx1 that disrupted its binding to 60S also suppressed an rei1delta mutant and restored the normal nuclear localization of Arx1. These results indicate that the cold sensitivity of rei1delta cells is due to the persistence of Arx1 on 60S subunits in the cytoplasm. Furthermore, these results suggest that Rei1 is needed for release of Arx1 from nascent 60S subunits after export to the cytoplasm but not for the subsequent nuclear import of Arx1.