Long non-coding RNA Xist regulates oocyte loss via suppressing miR-23b-3p/miR-29a-3p maturation and upregulating STX17 in perinatal mouse ovaries.

The fecundity of female mammals is resolved by the limited size of the primordial follicle (PF) pool formed perinatally. The establishment of PF pool is accompanied by a significant programmed oocyte death. Long non-coding RNAs (lncRNA) are central modulators in regulating cell apoptosis or autophagy in multiple diseases, however, the significance of lncRNAs governing perinatal oocyte loss remains unknown. Here we find that Yin-Yang 1 (YY1) directly binds to the lncRNA X-inactive-specific transcript (Xist) promoter and facilitates Xist expression in the perinatal mouse ovaries. Xist is highly expressed in fetal ovaries and sharply downregulated along with the establishment of PF pool after birth. Gain or loss of function analysis reveals that Xist accelerates oocyte autophagy, mainly through binding to pre-miR-23b or pre-miR-29a in the nucleus and preventing the export of pre-miR-23b/pre-miR-29a to the cytoplasm, thus resulting in decreased mature of miR-23b-3p/miR-29a-3p expression and upregulation miR-23b-3p/miR-29a-3p co-target, STX17, which is essential for timely control of the degree of oocyte death in prenatal mouse ovaries. Overall, these findings identify Xist as a key non-protein factor that can control the biogenesis of miR-23b-3p/miR-29a-3p, and this YY1-Xist-miR-23b-3p/miR-29a-3p-STX17 regulatory axis is responsible for perinatal oocyte loss through autophagy.

Snord116 Post-transcriptionally Increases Nhlh2 mRNA Stability: Implications for Human Prader-Willi Syndrome.

The smallest genomic region causing Prader-Willi Syndrome (PWS) deletes the non-coding RNA SNORD116 cluster; however, the function of SNORD116 remains a mystery. Previous work in the field revealed the tantalizing possibility that expression of NHLH2, a gene previously implicated in both obesity and hypogonadism, was downregulated in PWS patients and differentiated stem cells. In silico RNA: RNA modeling identified several potential interaction domains between SNORD116 and NHLH2 mRNA. One of these interaction domains was highly conserved in most vertebrate NHLH2 mRNAs examined. A construct containing the Nhlh2 mRNA, including its 3'-UTR, linked to a c-myc tag was transfected into a hypothalamic neuron cell line in the presence and absence of exogenously-expressed Snord116. Nhlh2 mRNA expression was upregulated in the presence of Snord116 dependent on the length and type of 3'UTR used on the construct. Furthermore, use of actinomycin D to stop new transcription in N29/2 cells demonstrated that the upregulation occurred through increased stability of the Nhlh2 mRNA in the 45 minutes immediately following transcription. In silico modeling also revealed that a single nucleotide variant (SNV) in the NHLH2 mRNA could reduce the predicted interaction strength of the NHLH2:SNORD116 diad. Indeed, use of an Nhlh2 mRNA construct containing this SNV significantly reduces the ability of Snord116 to increase Nhlh2 mRNA levels. For the first time, these data identify a motif and mechanism for SNORD116-mediated regulation of NHLH2, clarifying the mechanism by which deletion of the SNORD116 snoRNAs locus leads to PWS phenotypes.

A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish.

Cytoplasmic male sterility (CMS) observed in many plants leads defect in the production of functional pollen, while the expression of CMS is suppressed by a fertility restorer gene in the nuclear genome. Ogura CMS of radish is induced by a mitochondrial orf138, and a fertility restorer gene, Rfo, encodes a P-type PPR protein, ORF687, acting at the translational level. But, the exact function of ORF687 is still unclear. We found a Japanese variety showing male sterility even in the presence of Rfo. We examined the pollen fertility, Rfo expression, and orf138 mRNA in progenies of this variety. The progeny with Type H orf138 and Rfo showed male sterility when their orf138 mRNA was unprocessed within the coding region. By contrast, all progeny with Type A orf138 were fertile though orf138 mRNA remained unprocessed in the coding region, demonstrating that ORF687 functions on Type A but not on Type H. In silico analysis suggested a specific binding site of ORF687 in the coding region, not the 5' untranslated region estimated previously, of Type A. A single nucleotide substitution in the putative binding site diminishes affinity of ORF687 in Type H and is most likely the cause of the ineffectiveness of ORF687. Furthermore, fertility restoration by RNA processing at a novel site in some progeny plants indicated a new and the third fertility restorer gene, Rfs, for orf138. This study clarified that direct ORF687 binding to the coding region of orf138 is essential for fertility restoration by Rfo.

LC-MS/MS Profiling of Post-Transcriptional Modifications in Ginseng tRNA Purified by a Polysaccharase-Aided Extraction Method.

Transfer RNAs (tRNAs) are the most heavily modified RNA species in life entities. Post-transcriptional modifications severely impact the structure and function of tRNAs. To date, hundreds of modifications have been identified in tRNAs, mainly from microorganisms and animals. However, tRNAs in plant roots or tubers that have been widely used for food and medical purpose for centuries are rarely studied because isolation of RNA from plants still remains a challenge. In this paper, a polysaccharase-aided RNA isolation (PARI) method for extraction of high-quality RNA from plants containing large quantities of polysaccharides is developed. This method presents a new strategy of "digesting" polysaccharides that is completely different from the conventional method of "dissolving" the contaminants. By using this method, RNA of high integrity and purity were successfully extracted from ginseng roots because polysaccharide contaminations were removed efficiently with α-amylase digestion. Ginseng tRNAs were first sequenced by NGS and a total of 41 iso acceptors were identified. ChloroplastictRNAGly(GCC) in ginseng root was purified and four modified nucleosides, including m7G, D, T, and Ψ, were identified by LC-MS/MS. The results also revealed that the m7G occurs at a novel position 18, which may be related to the deformation of D-loop. PARI is the first enzyme-assisted technique for RNA isolation from plants, which could fundamentally solve the problem of polysaccharide contaminations. By using the PARI method, more individual tRNAs could be isolated easily from polysaccharide-rich plant tissues, which would have a positive impact on the feasibility of research on structure and function of tRNA in plants.

A comparative proteomic analysis of Pinellia ternata leaves exposed to heat stress.

Pinellia ternata is an important traditional Chinese medicinal plant. The growth of P. ternata is sensitive to high temperatures. To gain a better understanding of heat stress responses in P. ternata, we performed a comparative proteomic analysis. P. ternata seedlings were subjected to a temperature of 38 °C and samples were collected 24 h after treatment. Increased relative ion leakage and lipid peroxidation suggested that oxidative stress was frequently generated in rice leaves exposed to high temperature. Two-dimensional electrophoresis (2-DE) was used to analyze heat-responsive proteins. More than 600 protein spots were reproducibly detected on each gel; of these spots, 20 were up-regulated, and 7 were down-regulated. A total of 24 proteins and protein species were successfully identified by MALDI-TOF/TOF MS. These proteins and protein species were found to be primarily small heat shock proteins (58%) as well as proteins involved in RNA processing (17%), photosynthesis (13%), chlorophyll biosynthetic processes (4%), protein degradation (4%) and defense (4%). Using 2-DE Western blot analysis, we confirmed the identities of the cytosolic class II small heat shock protein (sHSPs-CII) identified by MS. The expression levels of four different proteins [cytosolic class I small heat shock protein (sHSPs-CI), sHSPs-CII, mitochondrial small heat shock protein (sHSPs-MIT), glycine-rich RNA-binding protein (GRP)] were analyzed at the transcriptional level by quantitative real-time PCR. The mRNA levels of three sHSPs correlated with the corresponding protein levels. However, GRP was down-regulated at the beginning of heat stress but then increased substantially to reach a peak after 24 h of heat stress. Our study provides valuable new insight into the responses of P. ternata to heat stress.

Expression and post-transcriptional regulation of maize transposable element MuDR and its derivatives.

The transposition of Mu elements underlying Mutator activity in maize requires a transcriptionally active MuDR element. Despite variation in MuDR copy number and RNA levels in Mutator lines, transposition events are consistently late in plant development, and Mu excision frequencies are similar. Here, we report previously unsuspected and ubiquitous MuDR homologs that produce both RNA and protein. MuDR transcript levels are proportional to MuDR copy number, and homolog transcript levels increase in active Mutator lines. A subset of homologs exhibits constitutive transcription in MuDR(-) and epigenetically silenced MuDR lines, suggesting independent transcriptional regulation. Surprisingly, immunodetection demonstrated nearly invariant levels of MuDR and homolog protein products in all tested Mutator and non-Mutator stocks. These results suggest a strict control over protein production, which might explain the uniform excision frequency of Mu elements. Moreover, the nonfunctional proteins encoded by homologs may negatively regulate Mutator activity and represent part of the host defense against this transposon family.

Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences.

Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.

Inactivation of the first nucleotide-binding fold of the sulfonylurea receptor, and familial persistent hyperinsulinemic hypoglycemia of infancy.

Familial persistent hyperinsulinemic hypoglycemia of infancy is a disorder of glucose homeostasis and is characterized by unregulated insulin secretion and profound hypoglycemia. Loss-of-function mutations in the second nucleotide-binding fold of the sulfonylurea receptor, a subunit of the pancreatic-islet beta-cell ATP-dependent potassium channel, has been demonstrated to be causative for persistent hyperinsulinemic hypoglycemia of infancy. We now describe three additional mutations in the first nucleotide-binding fold of the sulfonylurea-receptor gene. One point mutation disrupts the highly conserved Walker A motif of the first nucleotide-binding-fold region. The other two mutations occur in noncoding sequences required for RNA processing and are predicted to disrupt the normal splicing pathway of the sulfonylurea-receptor mRNA precursor. These data suggest that both nucleotide-binding-fold regions of the sulfonylurea receptor are required for normal regulation of beta-cell ATP-dependent potassium channel activity and insulin secretion.

Editing and import: strategies for providing plant mitochondria with a complete set of functional transfer RNAs.

The recombinations and mutations that plant mitochondrial DNA has undergone during evolution have led to the inactivation or complete loss of a number of the 'native' transfer RNA genes deriving from the genome of the ancestral endosymbiont. Following sequence divergence in their genes, some native mitochondrial tRNAs are 'rescued' by editing, a post-transcriptional process which changes the RNA primary sequence. According to in vitro studies with the native mitochondrial tRNA(Phe) from potato and tRNA(His) from larch, editing is required for efficient processing. Some of the native tRNA genes which have been inactivated or lost have been replaced by tRNA genes present in plastid DNA sequences acquired by the mitochondrial genome during evolution, which raises the problem of the transcriptional regulation of tRNA genes in plant mitochondria. Finally, tRNAs for which no gene is present in the mitochondrial genome are imported from the cytosol. This process is highly specific for certain tRNAs, and it has been suggested that the cognate aminoacyl-tRNA synthetases may be responsible for this specificity. Indeed, a mutation which blocks recognition of the cytosolic Arabidopsis thaliana tRNA(Ala) by the corresponding alanyl-tRNA synthetase also prevents mitochondrial import of this tRNA in transgenic plants. Conversely, no significant mitochondrial co-import of the normally cytosol-specific tRNA(Asp) was detected in transgenic plants expressing the yeast cytosolic aspartyl-tRNA synthetase fused to a mitochondrial targeting sequence, suggesting that, although necessary, recognition by a cognate aminoacyl-tRNA synthetase might not be sufficient to allow tRNA import into plant mitochondria.

Cloning and characterisation of a U6 small nuclear RNA gene from potato.

Using a mixed U6 snRNA gene probe and a low stringency hybridization procedure we have isolated a U6 snRNA containing clone from a potato genomic library in lambda EMBL 3. This clone contains a single U6 snRNA gene which has been subcloned and sequenced. Southern blotting experiments using this gene and the heterologous U6 genes as probes indicate that the potato U6 gene family consists of more than 20 members. The potato U6 gene sequence shows high identity to previously characterised plant U6 snRNA gene sequences and possesses correctly positioned and spaced transcription control elements suggesting that it is an active gene.

Sequence variation and linkage of potato U2snRNA-encoding genes established by PCR.

Plant uridylate-rich small nuclear RNA (UsnRNA)-encoding genes (UsnRNA) are present as multigene families exhibiting greater sequence variation than has been described in animal UsnRNA families. The potato U2snRNA multigene family has 25 to 40 potential gene members. Four gene variants have been analysed to date, two of which are linked. In order to investigate U2snRNA expression in potato in terms of the function of such sequence variation in development, the degree of sequence variation in both the coding region and flanking regions in this gene family must be assessed. On the assumption that at least some U2snRNA genes are linked, a polymerase chain reaction (PCR) approach, using primers designed to amplify intergenic nucleotide sequences including coding and 5' flanking regions, has been devised. Six new U2snRNA gene variant sequences and one U2snRNA pseudogene sequence have been generated. In addition, six new flanking region sequences have been produced which, in contrast to other plant UsnRNA gene families, show considerable variation in the important upstream sequence element. This PCR approach may be applicable to the analysis of genomic organisation and sequence variation of other multigene families.