Long noncoding RNA SPRY4-IT1 regulates intestinal epithelial barrier function by modulating the expression levels of tight junction proteins

Epithelial cells line the intestinal mucosa and form an important barrier to a wide array of noxious substances in the lumen. Disruption of the barrier integrity occurs commonly in various pathologies. Long noncoding RNAs (lncRNAs) control diverse biological processes, but little is known about the role of lncRNAs in regulation of the gut permeability. Here we show that the lncRNA SPRY4-IT1 regulates the intestinal epithelial barrier function by altering expression of tight junction (TJ) proteins. SPRY4-IT1 silencing led to dysfunction of the epithelial barrier in cultured cells by decreasing the stability of mRNAs encoding TJ proteins claudin-1, claudin-3, occludin, and JAM-1 and repressing their translation. In contrast, increasing the levels of SPRY4-IT1 in the intestinal mucosa protected the gut barrier in mice exposed to septic stress by increasing the abundance of TJ proteins. SPRY4-IT1 directly interacted with TJ mRNAs, and this process was enhanced through the association with the RNA-binding protein HuR. Of interest, the intestinal mucosa from patients with increased gut permeability exhibited a decrease in the levels of SPRY4-IT1. These findings highlight a novel role for SPRY4-IT1 in controlling the intestinal epithelial barrier and define a mechanism by which SPRY4-IT1 modulates TJ expression by altering the stability and translation of TJ mRNAs.     

Functional characterization of a geraniol synthase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> and its application in production of geraniol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Geraniol synthase (GES) catalyzes the conversion of geranyl diphosphate (GPP) into geraniol, an acyclic monoterpene alcohol that has been widely used in many industries. Here we report the functional characterization of CaGES from Camptotheca acuminata, a camptothecin-producing plant, and its application in production of geraniol in Escherichia coli. The full-length cDNA of CaGES was obtained from overlap extension PCR amplification. The intact and N-terminus-truncated CaGESs were overexpressed in E. coli and purified to homogeneity. Recombinant CaGES showed the conversion activity from GPP to geraniol. To produce geraniol in E. coli using tCaGES, the biosynthetic precursor GPP should be supplied and transferred to the catalytic pocket of tCaGES. Thus, ispA(S80F), a mutant of farnesyl diphosphate (FPP) synthase, was prepared to produce GPP via the head-to-tail condensation of isoprenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A slight increase of geraniol production was observed in the fermentation broth of the recombinant E. coli harboring tCaGES and ispA(S80F). To enhance the supply of IPP and DMAPP, the encoding genes involved in the whole mevalonic acid biosynthetic pathway were introduced to the E. coli harboring tCaGES and the ispA(S80F) and a significant increase of geraniol yield was observed. The geraniol production was enhanced to 5.85 ± 0.46 mg L^?1 when another copy of ispA(S80F) was introduced to the above recombinant strain. The following optimization of medium composition, fermentation time, and addition of metal ions led to the geraniol production of 48.5 ± 0.9 mg L^?1. The present study will be helpful to uncover the biosynthetic enigma of camptothecin and tCaGES will be an alternative to selectively produce geraniol in E. coli with other metabolic engineering approaches.     

Somaclonal variation does not preclude the use of rice transformants for genetic screening

Rice (Oryza sativa) is one of the world's most important crops. Rice researchers make extensive use of insertional mutants for the study of gene function. Approximately half a million flanking sequence tags from rice insertional mutant libraries are publicly available. However, the relationship between genotype and phenotype is very weak. Transgenic plant assays have been used frequently for complementation, overexpression or antisense analysis, but sequence changes caused by callus growth, Agrobacterium incubation medium, virulence genes, transformation and selection conditions are unknown. We used high‐throughput sequencing of DNA from rice lines derived from Tainung 67 to analyze non‐transformed and transgenic rice plants for mutations caused by these parameters. For comparison, we also analyzed sequence changes for two additional rice varieties and four T‐DNA tagged transformants from the Taiwan Rice Insertional Mutant resource. We identified single‐nucleotide polymorphisms, small indels, large deletions, chromosome doubling and chromosome translocations in these lines. Using standard rice regeneration/transformation procedures, the mutation rates of regenerants and transformants were relatively low, with no significant differences among eight tested treatments in the Tainung 67 background and in the cultivars Taikeng 9 and IR64. Thus, we could not conclusively detect sequence changes resulting from Agrobacterium‐mediated transformation in addition to those caused by tissue culture‐induced somaclonal variation. However, the mutation frequencies within the two publically available tagged mutant populations, including TRIM transformants or Tos17 lines, were about 10‐fold higher than the frequency of standard transformants, probably because mass production of embryogenic calli and longer callus growth periods were required to generate these large libraries.