The proteasome regulatory particle (RP) initiates the degradation of ubiquitin-protein conjugates by unfolding the substrate and translocating it into the proteasome core particle (CP) to be degraded1. Here we show that in yeast three proteins are found associated with RP but not RP-CP holoenzyme: Nas6, Rpn14, and Hsm3. Mutations in these genes confer proteasome loss of function phenotypes, despite their virtual absence from holoenzyme. These effects result from deficient RP assembly. Thus, Nas6, Rpn14, and Hsm3 are RP chaperones. The RP contains six ATPases–the Rpt proteins–and each RP chaperone binds to the C-terminal domain of a specific Rpt. We show in an accompanying study2 that RP assembly is templated through the Rpt C-termini, apparently by their insertion into binding pockets in the CP. Thus, RP chaperones may regulate proteasome assembly by directly restricting the accessibility of Rpt C-termini to the CP. In addition, competition between the CP and RP chaperones for Rpt engagement may explain the release of RP chaperones as proteasomes mature.

Ripening of the model fruit tomato (Solanum lycopersicum) is controlled by a transcription factor network including NAC (NAM, ATAF1/2, and CUC2) domain proteins such as No-ripening (NOR), SlNAC1, and SlNAC4, but very little is known about the NAC targets or how they regulate ripening. Here, we conducted a systematic search of fruit-expressed NAC genes and showed that silencing NOR-like1 (Solyc07g063420) using virus-induced gene silencing (VIGS) inhibited specific aspects of ripening. Ripening initiation was delayed by 14 days when NOR-like1 function was inactivated by CRISPR/Cas9 and fruits showed obviously reduced ethylene production, retarded softening and chlorophyll loss, and reduced lycopene accumulation. RNA-sequencing profiling and gene promoter analysis suggested that genes involved in ethylene biosynthesis (SlACS2, SlACS4), color formation (SlGgpps2, SlSGR1), and cell wall metabolism (SlPG2a, SlPL, SlCEL2, and SlEXP1) are direct targets of NOR-like1. Electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), and dual-luciferase reporter assay (DLR) confirmed that NOR-like1 bound to the promoters of these genes both in vitro and in vivo, and activated their expression. Our findings demonstrate that NOR-like1 is a new positive regulator of tomato fruit ripening, with an important role in the transcriptional regulatory network.

AC133 is a member of a novel family of cell surface proteins with 5 transmembrane domains. The function of AC133 is unknown. Although AC133 mRNA is detected in different tissues, its expression in the hematopoietic system is restricted to CD34 ؉ stem cells. AC133 is also expressed on stem cells of other tissues, including endothelial progenitor cells. However, despite the potential importance of AC133 to the field of stem cell biology, nothing is known about the transcriptional regulation of AC133 expression. In this report we showed that the human AC133 gene has at least 9 distinctive 5-untranslated region (UTR) exons, resulting in the formation of at least 7 alternatively spliced 5-UTR isoforms of AC133 mRNA, which are expressed in a tissue-dependent manner. We found that transcription of these AC133 isoforms is controlled by 5 alternative promoters, and we demonstrated their activity on AC133-expressing cell lines using a luciferase reporter system. We also showed that in vitro methylation of 2 of these AC133 promoters completely suppresses their activity, suggesting that methylation plays a role in their regulation. Identification of tissue-specific AC133 promoters may provide a novel method to isolate tissuespecific stem and progenitor cells. (Blood.

The tomato non-ripening (nor) mutant generates a truncated 186-amino-acid protein (NOR186) and has been demonstrated previously to be a gain-of-function mutant. Here, we provide more evidence to support this view and answer the open question of whether the NAC-NOR gene is important in fruit ripening. Overexpression of NAC-NOR in the nor mutant did not restore the full ripening phenotype. Further analysis showed that the truncated NOR186 protein is located in the nucleus and binds to but does not activate the promoters of 1-aminocyclopropane-1-carboxylic acid synthase2 (SlACS2), geranylgeranyl diphosphate synthase2 (SlGgpps2), and pectate lyase (SlPL), which are involved in ethylene biosynthesis, carotenoid accumulation, and fruit softening, respectively. The activation of the promoters by the wild-type NOR protein can be inhibited by the mutant NOR186 protein. On the other hand, ethylene synthesis, carotenoid accumulation, and fruit softening were significantly inhibited in CR-NOR (CRISPR/Cas9-edited NAC-NOR) fruit compared with the wild-type, but much less severely affected than in the nor mutant, while they were accelerated in OE-NOR (overexpressed NAC-NOR) fruit. These data further indicated that nor is a gain-of-function mutation and NAC-NOR plays a significant role in ripening of wild-type fruit.

Increasing grain yield is the most important object of crop breeding. Here, we report that the elevated expression of a conserved microRNA, OsmiR408, could positively regulate grain yield in rice () by increasing panicle branches and grain number. We further showed that OsmiR408 regulates grain yield by down-regulating its downstream target, , which is an uclacyanin (UCL) gene of the phytocyanin family. The knock down or knock out of also increases grain yield, while the overexpression of results in an opposite phenotype. Spatial and temporal expression analyses showed that was highly expressed in pistils, young panicles, developing seeds, and inflorescence meristem and was nearly complementary to that of OsmiR408. Interestingly, the OsUCL8 protein was localized to the cytoplasm, distinct from a majority of phytocyanins, which localize to the plasma membrane. Further studies revealed that the cleavage of by miR408 affects copper homeostasis in the plant cell, which, in turn, affects the abundance of plastocyanin proteins and photosynthesis in rice. To our knowledge, this is the first report of the effects of miR408- in regulating rice photosynthesis and grain yield. Our study further broadens the perspective of microRNAs and UCLs and provides important information for breeding high-yielding crops through genetic engineering.