ADAR-mediated RNA editing in non-coding RNA sequences

Adenosine to inosine (A-to-I) RNA editing is the most abundant editing event in animals. It converts adenosine to inosine in double-stranded RNA regions through the action of the adenosine deaminase acting on RNA (ADAR) proteins. Editing of pre-mRNA coding regions can alter the protein codon and increase functional diversity. However, most of the A-to-I editing sites occur in the non-coding regions of pre-mRNA or mRNA and non-coding RNAs. Untranslated regions (UTRs) and introns are located in pre-mRNA non-coding regions, thus A-to-I editing can influence gene expression by nuclear retention, degradation, alternative splicing, and translation regulation. Non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA) and long non-coding RNA (lncRNA) are related to pre-mRNA splicing, translation, and gene regulation. A-to-I editing could therefore affect the stability, biogenesis, and target recognition of non-coding RNAs. Finally, it may influence the function of non-coding RNAs, resulting in regulation of gene expression. This review focuses on the function of ADAR-mediated RNA editing on mRNA non-coding regions (UTRs and introns) and non-coding RNAs (miRNA, siRNA, and lncRNA).     

Apoptotic Endonuclease EndoG Induces Alternative Splicing of Telomerase TERT Catalytic Subunit,Caspase-2, DNase I,and BCL-x in Human,Murine, and Rat CD4<Superscript>+</Superscript> T Lymphocytes

Apoptotic endonuclease EndoG plays a key role in the alternative splicing of mRNA of human TERT telomerase catalytic subunit. The aim of this work was to test the ability of EndoG to induce alternative splicing of mRNA of other genes and in other organisms. To determine new mRNA splice-variants, EndoG overexpression was induced in human, mouse and rat CD4⁺-T-lymphocytes followed by sequencing of total RNA of these cells. Sequencing results showed that besides TERT, EndoG induced alternative splicing of deoxyribonuclease I (DNase I), caspase-2 (Casp-2) and BCL-x. The expression level of EndoG strongly correlated with mRNA splicing-variants of TERT, DNase I, Casp-2, and BCL-x in intact CD4⁺-T cells of healthy donors as well as different lines of mice and rats. EndoG overexpression induced down-regulation of fulllength mRNAs of TERT, DNase I, Casp-2, and BCL-x and up-regulation of their short-length mRNAs. Alternative splicing of studied mRNAs resulted in down-regulation of enzymatic activity of proteins in vitro and in vivo. The results of this work confirm the ability of endonuclease EndoG to induce alternative splicing of several mRNAs in human, mice and rats.     

RNA splicing: disease and therapy

The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. The mechanism of pre-mRNA splicing is highly complex, requiring multiple interactions between pre-mRNA, small nuclear ribonucleoproteins and splicing factor proteins. Regulation of this process is even more complicated, relying on loosely defined cis-acting regulatory sequence elements, trans-acting protein factors and cellular responses to varying environmental conditions. Many different human diseases can be caused by errors in RNA splicing or its regulation. Targeting aberrant RNA provides an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. Antisense oligonucleotide therapies show particular promise in this area and, if coupled with improved delivery strategies, could open the door to a multitude of novel personalized therapies.     

人端粒酶逆转录酶(hTERT)基因第3内含子的克隆和分析

从端粒酶活性呈阳性的永生细胞株人肺腺癌细胞SPC A 1中分离了总RNA ,以此为模板 ,结合RT PCR技术和长模板PCR技术 ,用hTERT基因特异性引物扩增到一长约 2 .2kb的cDNA片段。将该片段纯化后克隆到通用测序载体T easyvector上得到重组质粒。用测序引物SP6和T7对该片段进行部分双向测序。经序列分析和同源比较推测该片段包含了hTERT基因的第 3内含子。该结果提示了RT PCR技术和长模板PCR技术用于真核生物基因内含子克隆的可行性。进一步的分析表明 ,该片段在不同细胞的RT PCR产物中的产量不同 ,提示hTERT基因前体mRNA中的第 3内含子可能在不同细胞中有不同的剪接效率。     

Regulation of protein diversity by alternative pre-mRNA splicing with specific focus on chondrogenesis

Analysis of the human genome has dramatically demonstrated that the majority of protein diversity is generated by alternative splicing of pre-mRNA. This powerful and versatile mechanism controls the synthesis of functionally different protein isoforms that may be required during specific stages of development from a single gene. Consequently, ubiquitous and/or tissue-specific RNA splicing factors that regulate this splicing mechanism provide the basis for defining phenotypic characteristics of cells during differentiation. In this review, we will introduce the basic mechanisms of pre-mRNA alternative splicing, describe how this process is regulated by specific RNA splicing factors, and relate this to various systems of cell differentiation. Chondrogenesis, a well-defined differentiation pathway necessary for skeletogenesis, will be discussed in detail, with focus on some of the alternatively-spliced proteins known to be expressed during cartilage development. We propose a heuristic view that, ultimately, it is the regulation of these RNA splicing factors that determines the differentiation status of a cell. Studying regulation at the level of pre-mRNA alternative splicing will provide invaluable insights into how many developmental mechanisms are controlled, thus enabling us to manipulate a system to select for a specific differentiation pathway.