Vitamin D3 correlates inversely with systemic dendritic cell numbers and bone erosion in chronic rhinosinusitis with nasal polyps and allergic fungal rhinosinusitis.

Vitamin D(3) (VD(3) ) is a steroid hormone that regulates bone health and numerous aspects of immune function and may play a role in respiratory health. We hypothesized that T helper type 2 (Th2) disorders, chronic rhinosinusitis with nasal polyps (CRSwNP) and allergic fungal rhinosinusitis (AFRS) would have VD(3) deficiencies, resulting in increased mature dendritic cells (DCs) and bone erosion. We conducted a retrospective study examining VD(3) levels in patients with AFRS (n = 14), CRSwNP (n = 9), chronic rhinosinusitis without nasal polyps (CRSsNP) (n = 20) and cerebrospinal fluid leak repair (non-diseased controls) (n = 14) at time of surgery. Circulating immune cell levels were determined by immunostaining and flow cytometric analysis. Plasma VD(3) and immune regulatory factors (granulocyte-macrophage colony-stimulating factor and prostaglandin E(2) ) were measured by enzyme-linked immunosorbent assay. It was observed that CRSwNP and AFRS demonstrated increased circulating DCs, while chronic rhinosinusitis without nasal polyps displayed increased circulating macrophages. CRSwNP and AFRS were to found to have insufficient levels of VD(3) which correlated inversely with circulating numbers of mature DCs, DC regulatory factors and bone erosion. CRSsNP displayed no change in circulating DC numbers or VD(3) status compared to control, but did display increased numbers of circulating macrophages that was independent of VD(3) status. Lastly, VD(3) deficiency was associated with more severe bone erosion. Taken together, these results suggest support a role for VD(3) as a key player in the immunopathology of CRSwNP and AFRS.

Heat stress results in incomplete C-to-U editing of maize chloroplast mRNAs and correlates with changes in chloroplast transcription rate.

The editing status rps14 and rpl20 mRNAs decreased rapidly from nearly 100% edited to about 30% edited when maize plants were shifted from 20 degrees C to 37 degrees C. A decrease in the extent of editing was easily detected within 2 h and decreased to a steady-state level of about 40% C-to-U conversion at 37 degrees C. In contrast, the editing status of these chloroplast mRNAs increased relatively slowly after plants were shifted from 37 degrees C to 20 degrees C. Chloroplasts isolated from maize plants which were grown at 20 degrees C and then shifted to 37 degrees C for 24 h were 5-10 times more transcriptionally active than chloroplasts isolated from maize plants grown continuously at 20 degrees C. Thus, the high transcription rate at 37 degrees C may establish a kinetic condition where the rate of transcription exceeds the capacity of the editing apparatus and results in incomplete editing.

Addition of non-genomically encoded nucleotides to the 3'-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA.

The 3'-termini of maize mitochondrial RNAs were characterized by ligation of an anchor oligonucleotide, reverse transcription and amplification. DNA sequence analysis of cDNA clones for tRNA(Ser) and 18S rRNA confirmed the expected 3'-terminal nucleotides and demonstrated the accuracy and fidelity of the protocol. Analysis of cDNAs for rps12, cox2 and atp9 indicated that non-genomically encoded nucleotides were present at the 3'-terminus. rps12 cDNAs exhibited the highest degree of modification, with 94% of 35 cDNA clones analyzed containing one to four non-genomically encoded C or A residues; 83% of these cDNAs terminated with the trinucleotide CCA. DNA sequence and transcript mapping analyses demonstrated that four positions exhibited modified 3'-termini within a small region of the 3' flank of rps12 transcripts. These transcript termini represented low abundance, truncated forms of rps12 mRNAs which may be intermediates in degradation. cox2 mRNAs are also modified at a truncated position. Sixty percent of the cox2 cDNAs were modified with 1-5 nt that most frequently included A and C residues, but also included a few G and T residues. Non-genomically encoded nucleotides were detected in 27% of the atp9 cDNAs as a single C or A residue.

Rubisco small and large subunit N-methyltransferases. Bi- and mono-functional methyltransferases that methylate the small and large subunits of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the alpha-amino group of the N-terminal methionine of the processed form of the small subunit (SS), and at the epsilon-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit (alpha)N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both (alpha)N-methylation of the SS and (epsilon)N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the alpha-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.

RNA editing site recognition in higher plant mitochondria.

RNA editing is a process by which genomically encoded cytidines are converted to uridines in plant mitochondrial transcripts. This conversion usually changes the amino acid specified by a codon and converts an "aberrant" residue to the evolutionarily conserved amino acid. The selection of the edited cytidine is highly specific. The cis-acting sequences for editing site recognition have been examined in ribosomal protein S12 (rps12) transcripts and in transcripts for a second copy of an internal portion of the ribosomal protein S12 (rps12b). rps12b was created by recombination at 7 and 9 nucleotide sequences that included editing sites I and IV of rps12, thus affording an opportunity to study the editing of chimeric transcripts with rearrangement very near C to U editing sites. Rearrangements downstream of editing site IV did not affect the editing of that sequence, while rearrangement upstream of editing site I ablated editing at that cytidine residue. Secondary structure predictions indicated that RNA structure did not correlate with the editing of these substrates. These results taken together with other studies in the literature suggest that RNA editing site recognition is primarily dependent on the 5' flanking RNA sequence.

Editing and translation of ribosomal protein S13 transcripts: unedited translation products are not detectable in maize mitochondria.

Maize mitochondrial transcripts for the ribosomal protein S13 gene (rps13) have six C- to -U editing sites, and each nucleotide conversion causes a change in the amino acid specified by the effected codons. Sequence analysis of 30 cDNA clones indicated that 73% of the cDNAS were edited at all six sites and 3% were completely unedited. Antibodies were produced against synthetic peptides that corresponded to unedited or edited translation products at editing sites V and VI (80% and 83% edited, respectively). Antibody preparations were purified that selectively recognized the edited or unedited forms of the epitope. The antibody preparations were highly sensitive to the amino-acid residue encoded at editing site VI, but relatively insensitive to the residue encoded at editing site V. Immunological analyses demonstrated that the edited translation product accumulated as a ribosomal protein, but that the unedited translation product was not detected in the mitochondrion, in the ribosomal fraction, or in a post-ribosomal supernatant. These results, taken together with other studies which demonstrated that incompletely edited transcripts are incorporated into polyribosomes, suggest that incompletely edited transcripts may be translated, but polypeptides encoded by incompletely edited RNAs may be unstable and, consequently, fail to accumulate.

Editing site recognition in plant mitochondria: the importance of 5'-flanking sequences.

Cytidine to uridine (C-to-U) editing occurs in plant mitochondria with very high specificity such that only specific cytidines are converted to uridines. The mechanisms for editing site selection in plant mitochondria are unknown. In order to examine the determinants of editing site recognition, repeated mitochondrial DNA sequences that include edited nucleotides have been evaluated as editing substrates. During evolution the maize mitochondrial ribosomal protein subunit 12 (rps12) gene recombined with intron 1 of the ribosomal protein subunit 3 (rps3) gene and a region of the S1-like sequence of the 2.3 kb plasmid. These recombinations created a second copy of an internal portion of the rps12 gene, known as rps12b, which includes the first four editing sites of rps12 transcripts. The duplicated sequence extends seven nucleotides upstream of editing site 1 and six nucleotides downstream from editing site 4. The sequences of rps12 and rps12b are identical between these sites except for a single change at -5 from editing site 1. These modifications did not effect C-to-U conversion at editing sites 2, 3, or 4 in rps12b; however, no editing was detected at editing site 1 in rps12b cDNAs. Thus, the 5' recombination abolished editing at site I, while the 3' recombination modified the downstream RNA sequence, but did not effect editing at site IV. Secondary structure prediction suggests that changes in editing site recognition do not correlate with differences in secondary structures, and that primary RNA sequence may be responsible for editing site specification.

Related alphaN- and epsilonN-methyltransferases methylate the large and small subunits of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is methylated at the ax amino position of the N-terminal methionyl residue of the processed and assembled form of the small subunit (SS), and is also methylated in some species at the epsilon-amino group of lysine-14 in the large subunit (LS). The gene (rbcMT-S) and cDNAs for the SS alphaN-methyltransferase (SSMT) from spinach (Spinach oleracea) have been cloned, sequenced, and expressed. The gene is closely related to a previously characterized LS methyltransferase (Rubisco LSMT) cDNA from pea (Rubisco LSMT) and a Rubisco LSMT gene from tobacco. Sequence analysis of the cDNA and transcript mapping experiments demonstrate that the rbcMT-S pre-mRNAs experience alternative 3' splice site selection, such that mRNAs for a long form with a four amino acid insertion and a short form are expressed at approximately equal abundance. The coding sequence of spinach SSMT includes a putative targeting presequence with sequence identity at a plastid processing site. A N-terminal truncated form of spinach SSMT was expressed and purified from E. coli cells. Both long and short forms of the cDNAs were shown to catalyze methylation of the a amine of the N-terminal methionine of the SS of Rubisco.

Signaling in plants.

Higher plants are sessile organisms that perceive environmental cues such as light and chemical signals and respond by changing their morphologies. Signaling pathways utilize a complex network of interactions to orchestrate biochemical and physiological responses such as flowering, fruit ripening, germination, photosynthetic regulation, and shoot or root development. In this session, the mechanisms of signaling systems that trigger plant responses to light and to the gaseous hormone, ethylene, were discussed. These signals are first sensed by a receptor and transmitted to the nucleus by a complex network. A signal may be transmitted to the nucleus by any of several systems including GTP binding proteins (G proteins), which change activity upon GTP binding; protein kinase cascades, which sequentially phosphorylate and activate a series of proteins; and membrane ion channels, which change ionic characteristics of the cells. The signal is manifested in the nucleus as a change in the activity of DNA-binding proteins, which are transcription factors that specifically interact and modulate the regulatory regions of genes. Thus, detection of an environmental signal is transmitted through a transduction pathway, and changes in transcription factor activity may coordinate changes in the expression of a portfolio of genes to direct new developmental programs.

Identification of a 4.5S-like ribonucleoprotein in maize mitochondria.

Escherichia coli has a ribonucleoprotein complex that is composed of a 114 nucleotide 4.5S RNA and a 48 kDa polypeptide (P48) that has been demonstrated to function in translation and in the secretion of periplasmic polypeptides. A small RNA of approximately 220 nucleotides has been identified in maize mitochondria that includes sequence identity with the highly conserved domain of the bacterial 4.5S RNA. The transcript is mitochondrially encoded and maps to a region upstream of the gene for ATP synthase subunit I. The mitochondrial 4.5S-like RNA has 15 nucleotides of sequence identity with the highly conserved region of the bacterial 4.5S RNA. Sucrose density gradient centrifugation of a maize mitochondrial lysate demonstrated that the 4.5S RNA is a component of a high molecular weight complex under native conditions, and could be disrupted by phenol. Anti-P48 immune serum immuno-precipitated a mitochondrial protein of approximately 48 kDa, and RNA gel blot analysis of the immunoprecipitation reaction indicated that the 4.5S-like RNA co-immuno-precipitated with the 48 kDa polypeptide. The mitochondrial 4.5S ribonucleoprotein complex could function in translation or protein targeting.

Developmental- and tissue-specificity of RNA editing in mitochondria of suspension-cultured maize cells and seedlings.

C to U editing of apt9, nad3, and cox2 mRNAs was investigated in maize seedlings at various developmental stages as well as in suspension-cultured cells. Heterogeneity of mRNAs that result from incomplete editing was analyzed for each gene and from five tissues or developmental conditions. The editing status of approximately 30 cDNA clones was determined by digestion with a restriction enzyme that discriminates between unedited and edited DNA sequences. The atp9 and spliced cox2 cDNAs were essentially completely edited in all samples examined. Analysis of three editing sites of nad3 cDNAs indicated that incompletely edited cDNAs were detected in all tissues and treatments with a temporal increase in the overall editing status, from 50% at 3 days to about 75% at 7 days. These results indicate that incompletely edited mRNAs are prevalent for some plant mitochondrial genes, and can change with developmental or growth conditions.

Protein polymorphism generated by differential RNA editing of a plant mitochondrial rps12 gene.

The rps12 gene transcripts encoding mitochondrial ribosomal protein S12 are partially edited in petunia mitochondria. Different petunia lines were found vary in the extent of rps12 transcript editing. To test whether multiple forms of RPS12 proteins are produced in petunia mitochondria as a result of partial editing, we probed mitochondrial proteins with specific antibodies against edited and unedited forms of a 13-amino-acid RPS12 peptide spanning two amino acids affected by RNA editing. Both antibodies reacted with mitochondrial proteins at the expected size for RPS12 proteins. The amounts of unedited RPS12 protein in different petunia lines correlate with the abundance of unedited transcripts in these plants. Unedited rps12 translation products are also detected in other plant species, indicating that polymorphism in mitochondrial rps12 expression is widespread. Moreover, we show that RPS12 proteins recognized by both edited-specific and unedited-specific antibodies are present in a petunia mitochondrial ribosome fraction. These results demonstrate that partially edited transcripts can be translated and that the protein product can accumulate to detectable levels. Therefore, genes exhibiting incompletely edited transcripts can encode more than one gene product in plant mitochondria.

Incomplete editing of rps12 transcripts results in the synthesis of polymorphic polypeptides in plant mitochondria.

C-to-U editing causes specific nucleotide changes in plant mitochondrial nRNAs that are required for the restoration of the evolutionarily conserved amino acid sequence. Transcripts for the ribosomal protein S12 gene (rps12) have six C-to-U editing sites and are highly heterogeneous as a result of incomplete editing. immunological analysis demonstrated that unedited or partially edited transcripts as well as edited mRNAs are translated. The edited rps12 translation products accumulate as ribosomal subunits, but the unedited rps12 translation products are present as unassembled subunits and are not detected in the ribosomes. Thus, gene expression is polymorphic as a result of incomplete C-to-U editing, and aberrant polypeptides are present from the translation of these mRNAs. However, because only the edited translation products accumulate in mitochondrial ribosomes, the overall expression of rps12 is rendered coherent by the selection

RNA Editing in Plant Mitochondria: [alpha]-Phosphate Is Retained during C-to-U Conversion in mRNAs.

RNA editing in higher plant mitochondria frequently results in the post-transcriptional conversion of specific cytidine residues to uridine residues and infrequently results in the reverse conversion. The mechanisms by which this transition could occur are deamination or transamination of the amide at C-4 of cytosine, transglycosylation of the ribosyl residue, or deletion of a CMP residue and insertion of a UMP residue. Intact maize or petunia mitochondria were supplied with [alpha]-32P-CTP to radiolabel CMP residues in the nascent transcripts, and the fate of the [alpha]-phosphate was examined by digestion of the RNA to nucleotide monophosphates and analysis by two-dimensional chromatography. A small fraction of radioactivity comigrated with UMP on two different two-dimensional thin-layer chromatography systems, and the amount of radiolabeled UMP increased between l0-min and 2-hr incubations. The conversion of cytidine-to-uridine residues was detected in the highly edited mRNA fraction but was not detected in the rRNA fraction. Recovery of radiolabeled UMP residues suggests that the [alpha]-phosphate is retained during the editing reaction. These results are consistent with either deamination or transamination, or transglycosylation mechanisms for RNA editing.

Distribution of maize mitochondrial transcripts in polysomal RNA: evidence for non-selectivity in recruitment of mRNAs.

The distribution of maize mitochondrial transcripts in polysomal RNA fractions obtained from root tissue, shoot tissue, or isolated intact mitochondria was analyzed. The distribution of cox3 transcripts that differ in 5' untranslated RNA sequence was similar in total polysomal and total mitochondrial RNA fractions, suggesting that 5' heterogeneity does not affect recruitment of transcripts into the polysomal RNA. The distribution of spliced and unspliced cox2 transcripts was also analyzed in polysomes from total tissue or isolated mitochondria, and both precursor and mature mRNAs were present in the high-molecular-weight RNA fraction. These results suggest that ribosomal association with mitochondrial transcripts is not selective.

RNA editing intermediates of cox2 transcripts in maize mitochondria.

Eighteen cytidines are changed to uridines in the coding sequence of transcripts for cytochrome c oxidase subunit 2 (cox2) in maize mitochondria. The temporal relationship of editing and splicing was examined in cox2 transcripts by sequence analysis of spliced and unspliced cDNAs. Cloned cDNAs of unspliced cox2 transcripts ranged from clones with no edited nucleotides to completely edited forms, while spliced cDNAs were nearly completely edited. Incompletely edited transcripts in the nascent pool of unspliced transcripts represent intermediates of the editing process. These results indicate that editing proceeds without a strong directional bias and suggest that RNA editing is a posttranscriptional process.

Protection of tryptic-sensitive sites in the large subunit of ribulosebisphosphate carboxylase/oxygenase by catalysis.

Limited tryptic proteolysis of spinach (Spinacia oleracea) ribulose bisphosphate carboxylase/oxygenase (ribulose-P(2) carboxylase) resulted in the ordered release of two adjacent N-terminal peptides from the large subunit, and an irreversible, partial inactivation of catalysis. The two peptides were identified as the N-terminal tryptic peptide (acetylated Pro-3 to Lys-8) and the penultimate tryptic peptide (Ala-9 to Lys-14). Kinetic comparison of hydrolysis at Lys-8 and Lys-14, enzyme inactivation, and changes in the molecular weight of the large subunit, indicated that proteolysis at Lys-14 correlated with inactivation, while proteolysis at Lys-8 occurred much more rapidly. Thus, enzyme inactivation is primarily the result of proteolysis at Lys-14. Proteolysis of ribulose-P(2) carboxylase under catalytic conditions (in the presence of CO(2), Mg(2+), and ribulose-P(2)) also resulted in ordered release of these tryptic peptides; however, the rate of proteolysis at lysyl residues 8 and 14 was reduced to approximately one-third of the rate of proteolysis of these lysyl residues under noncatalytic conditions (in the presence of CO(2) and Mg(2+) only). The protection of these lysyl residues from proteolysis under catalytic conditions could reflect conformational changes in the N-terminal domain of the large subunit which occur during the catalytic cycle.

Transcriptional and posttranscriptional regulation of maize mitochondrial gene expression.

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of N epsilon-trimethylation.

Numerous transcription initiation sites exist for the maize mitochondrial genes for subunit 9 of the ATP synthase and subunit 3 of cytochrome oxidase.

Transcripts for plant mitochondrial genes are frequently present as multiple size classes. In maize, these differences often result from variation in the 5' noncoding region. To determine where transcription initiates, primary (unprocessed) transcripts were specifically labeled in vitro by the capping reaction catalyzed by guanylyltransferase. Direct mapping of transcription initiation sites was accomplished by hybridization of in vitro-capped RNA with the 5' flanking sequences of mitochondrial genes and subsequent digestion with single-strand-specific RNases. The RNase protection experiments identified three transcription initiation sites for subunit 3 of cytochrome oxidase and at least six transcription initiation sites for subunit 9 of ATP synthase. Thus, transcript size heterogeneity is primarily the result of multiple transcription initiation sites for these genes rather than RNA processing. Primer extension analyses of maize mitochondrial RNA were used to precisely establish the sequences at the initiation sites. Comparison of sequences at transcription initiation sites suggests that some homology exists at these sites, although no highly conserved consensus sequence is obvious.