| Non-coding RNAs (ncRNAs) are a class of RNAs which can not be translated into proteins and lack of obvious open reading frame (ORF), and widely exist in the transcriptome of human genome, accounting for more than 90% of the total transcripts. NcRNA can be generated from introns and intergenic sequences, or sometimes from the locus of coding gene by transcription from the opposite strand. Although we currently lack satisfactory classifications for these transcripts, long ncRNAs are arbitrarily considered to be longer than 200 nucleotides, for those shorter than 200nt are called short ncRNAs.NcRNAs were considered to be only the transcriptional "noise" of the genome. But the progress in RNA field in the past several years reveals that the expression of ncRNAs is temporally and spatially specific. And the identification of functional ncRNAs suggested that they may play very important roles in many cell processes. NcRNAs act functionaly as RNA in cells. They can exert their functions through base-pairing with other nucleic acids, simulating the structure of other molecules for competing binding, inhibiting the transcription of target genes through spatial constraints, changing epigenetic modification by recruiting chromatin remodeling complexes. So ncRNAs can regulate cell processes at multi-levels, including gene transcription, chromosome structure, RNA metabolism, mRNA stability and translation, and protein functions.Natural antisense transcripts (NATs) are RNA molecules which transcribed from the opposite DNA strand of other transcripts (sense RNA, usually are protein coding mRNA) and partly overlapped with sense RNAs. Most NATs belong to ncRNAs which can’t be translated into proteins in cell. NATs were first identified in viruses and prokaryotes, and following efforts revealed that the transcription of NATs is a common theme in eukaryotic cells. It is estimated that 70% transcript unit contain NATs in mouse and human genomes and about 17% of them are conserved between human and mouse. So far, only a minority of NATs have been identified and well studied. The big challenges we are facing today are to experimentally identify them and reveal their functions.Based on their genomic proximity to target genes, NATs can be classified into two categories, cis-NATs and trans-NATs (function out of their transcription locus). Normally, cis-NATs function in their transcription loci and contain relatively long overlapping regions with their corresponding sense mRNA. They usually regulate the expression of the sense mRNA in a "one to one" mode. In contrast, trans-NATs act at genomic loci different from its transcription region. And the complemetarity with its target RNAs are imperfect. So it can therefore regulate the expression of many target genes.Both sense and antisense RNAs can encode proteins or be non-protein-coding transcripts; however, the most prominent form of antisense transcription in the mammalian genome is a non-protein-coding antisense RNA partner of a protein-coding gene. The presence of non-protein-coding sense and antisense transcript pairs implies that natural antisense transcripts may also regulate non-protein-coding sense RNAs. NATs can regulate the expression of target genes in many ways, such as transcriptional collision, genomic rearrangements, chromatin remodeling, genomic imprinting, alternative splicing, transport, nuclear retention and mRNA editing, changes in mRNA stability and translation efficiency, masking miRNA-binding sites, formation of endogenous siRNA. Most of the identified NATs have been shown to downregulate the expression of their target genes. But there are also some cases in which the NATs upregulate the expression of their target genes.Based on the published literatures and bioinformatic analysis of the expressed sequence tag (EST) database in Genbank, we have identified a natural antisense transcript (TIRAP-AS) to TIRAP/MAL (TIR domain-containing adaptor protein/MyD88 adaptor-like), the adaptor protein downstream TLR2 and TLR4 receptors. It is conserved between mouse and human. In mouse, the genomic gene of TIRAP maps to chromosome 9 and consists of 7 exons with a full length about 16kb. And our identified TIRAP-AS contains 3 exons and the first exon is full complementary to part of the 6th exon of TIRAP gDNA. The similar genomic organization also found in human. And the splicing of TIRAP-AS RNA follows the GT-AG rule and TIRAP-AS gene contains canonical polyA signal. Furthermore, one of the identified TIRAPS-AS EST (UI-M-BH1-ami-a-04-0-UI.s2) contains polyA sequence. This suggests that TIRAP-AS RNA has been processed post its transcription and is not the products of non-specific transcription activities of cells.TIRAP (TIR domain-containing adaptor protein), also called Mal (MyD88-like adaptor), is a TIR domain-containing adaptor which functions in the MyD88-dependent pathway downstream TLR2 and TLR4 receptors. It firstly relocates to the plasma membrane after the activation of TLR2 or TLR4 receptor and binds to PIP2 through its PIP2 binding domain. Then it will recruit MyD88 to the the receptors and activate the downstream signaling pathways which will lead to the activation of downstream MAPK kinases such as p38MAPK and PI3K protein kinases and transcription factor NF-κB. That will induce the synthesis and release of proinflammatory cytokines such as TNF-a and IL-1. So TIRAP is indispensible in the MyD88-dependent pathway downstream TLR2 and TLR4 receptors.In a word, TIRAP plays an important role in the signal transduction pathway downstream TLR receptors. Functional dysregulation of TIRAP will directly lead to the innate immune abnormalities. At present, our knowledge of the expression mechanism of TIRAP gene is insufficiency. Does the antisense TIRAP-AS RNA regulate the expression of TIRAP? What is molecular regulation mechanism if the answer is yes? What is the functional implication of this regulation? Answers to these questions will be beneficial in deciphering the regulation mechanism of the expression TIRAP and promote our understanding of the function of TIRAP in innate immune responses.Based on the identified EST sequence of TIRAP-AS, we first cloned the full length of TIRAP-AS cDNA from RAW264.7 macrophage cell line by using RACE PCR. We have obtained 10 alternative splicing variants, ranging from 1000nt to 1200nt, of TIRAP-AS.The overlapping region between TIRAP-AS and TIRAP mRNA is 447nt. All of the variants lack obvious open reading frame. It suggests that TIRAP-AS functions as RNA in cells. The splicing of all TIRAP-AS variants follows the canonical GT-AG rule and the 3’RACE cDNA clones of TIRAP-AS contain polyA sequence. It means that TIRAP-AS is a fully processed RNA in cells.Then we used RT-PCR to analyze the expression of different splicing variants in RAW264.7 cell. And the cloned TIRAP-AS variants were used as positive control. It showed that the expression level of 4 splicing variants (1A, 1B,1D and 1E) were relatively higher than those of others. We also analyzed the relative expression of TIRAP mRNA to TIRAP-AS by using real-time PCR. The series diluted TIRAP and TIRAP-AS plasmids were used as standard. Our results reveal that the ratio of TIRAP mRNA to TIRAP-AS RNA level is about 8 to 1 in RAW264.7 cells.The expression level of TIRAP mRNA and TIRAP-AS in different tissue of mouse were also analyzed by using real-time PCR. The housekeeping gene β-actin was used as an internal control and normalization analysis. The expression level of TIRAP mRNA from high to low is liver, muscle, heart, kidney, lung, pancreas, skin and brain. In contrast, the sequence for TIRAP-AS RNA is liver, muscle, heart, lung, brain, skin, kidney and pancreas. And the expression level of TIRAP protein in different tissues was also analyzed by western blotting and the sequence for TIRAP protein is brain, heart, muscle, pancreas, lung, liver, skin, and kidney.It is slightly different from the expression level of TIRAP mRNA. This suggested that post-trancriptional regulation plays an important role in the expression of TIRAP protein.To analyze the effects of activation of TLR2 and TLR4 receptor on the expression of TIRAP mRNA and TIRPA-AS RNA, RAW264.7 cells were treated with 100ng/ml LPS (TLR4 specific agonist) and 100ng/ml Pam2CSK4 (TLR2 specific agonist) respectively. The cells were collected at different time (Omin,30min, 60min,120min,240min,480min) after the treatment. Then the mRNA level of TIRAP and TIRAP-AS were analyzed with Real-Time RT-PCR and the protein level of TIRAP was also analyzed by using Western Blotting. Our results revealed that activation of TLR2 or TLR4 led to time-dependent decrease of TIRAP-AS RNA. But the expression of TIRAP mRNA decreases quickly upon LPS treatment and then slowly backs to normal level. And the protein level of TIRAP in RAW264.7 cell upon LPS treatment also showed time-dependent decrease. The expression of TIRAP-AS RNA in BMM cells treated with 100ng/ml led to the similar changes as that of in RAW264.7 cells.At the same time, the effects of activation of other TLRs on the expression of TIRAP mRNA and TIRAP-AS RNA were also analyzed in RAW264.7 cells treated with mouse TLR1-9 agonists. The expression level of TIRAP mRNA and TIRAP-AS RNA significantly decreased in RAW264.7 cells treated with Pam3CSK4 (TLR1/2 specific agonist) and ODN1826 (TLR9 specific agonist) for 1h (p<0.05). These results suggest that the dynamic changes of TIRAP mRNA and TIRAP-AS RNA levels may be involved in the regulation of inflammatory reactions.To further analyze the mechanism of dynamic changes of TIRAP mRNA and TIRAP-AS RNA upon LPS treatment, RAW264.7 cells pretreated with specific kinase inhibitors, such as wortmannin (PI3K/Akt inhibitor), SP600125 (JNK inhibitor), PD98059 (ERK inhibitor) and SB203580(p38 inhibitor) for 1h were stimulated with LPS for 1h. Real-time PCR results showed that pretreatment with 200nM wortmannin specifically suppressed the deccrease of TIRAP-AS RNA induced by LPS treatment (p<0.05). It suggested that PI3K/Akt kinase activation is involved in the downregulation of TIRAP-AS RNA upon LPS treatment.The stability of TIRAP mRNA and TIRAP-AS RNA in RAW264.7 cell treated with LPS were also analyzed with real-time PCR. Our data revealed that LPS treatment promoted the degradation of TIRAP-AS RNA. But it had no effects on the mRNA stability of TIRAP.And pretreated with 200nM wortmannin suppressed the induced degradation of TIRAP-AS RNA upon LPS treatment. This suggested that activation of PI3K upon LPS treatment led to the enhanced degradation of TIRAP-AS RNA.In conclusion, we identified TIRAP-AS cDNA EST sequence by retrieving the NCBI database and bioinformatics analysis. The full-length of TIRAP-AS sequence was obtained by RACE PCR. Activation of TLR2 and TLR4 receptors promoted the degradation of TIRAP-AS through activation of PI3K/Akt kinase. But the molecular mechanism of activation of PI3K/Akt leads to the enhanced degradation of TIRAP-AS is not clear at present. |
PDF Full Text Request