The emerging role of circular RNAs in transcriptome regulation.

Circular RNAs (circRNAs) are a recently discovered form of RNA that has been found to regulate mammalian transcription. CircRNAs are covalently closed, single-stranded transcripts produced from precursor mRNA. While initially circRNAs were considered to be splicing artefacts, next-generation RNA sequencing of non-polyadenylated transcriptomes has recently shown that the expression of circRNAs is widespread and over 20% of expressed genes in examined cells and tissues can produce these transcripts. Until now thousands of circRNAs have been discovered in organisms ranging from Drosophila melanogaster to Homo sapiens. Functional studies indicate that these transcripts regulate expression of protein-coding linear transcripts and thus comprise an important component of gene expression regulation. Here we provide a comprehensive overview on the biology of circRNAs, including the expression patterns and function. Moreover, we discuss current methodologies for the discovery and validation of circular transcripts. Finally, perspectives on the utilization of circRNA as molecular markers of complex diseases are presented.

Strand-specific RNA-sequencing analysis of multiple system atrophy brain transcriptome.

Multiple system atrophy (MSA) is a sporadic neurodegenerative disease. The major pathological hallmark of MSA is the accumulation of α-synuclein in oligodendrocytes. In contrast to Parkinson's disease no definitive familial etiology for MSA has been determined. Yet, there is a growing body of evidence that perturbation of transcriptional processes leads to MSA pathology. Here we present the results of the first ribosomal-depleted strand-specific RNA-sequencing profile of the MSA brain frontal cortex tissue. Among the 123 differentially expressed genes over 50% were categorized as putative long intervening non-coding RNAs (lincRNAs). Along with the dysregulation of the non-coding portion of the transcriptome, the expression of protein coding genes was also affected, including serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (SERPINA3), interleukin 1 receptor-like 1 (IL1RL1) and hemoglobin, beta (HBB). Also of interest was the alternative splicing of SNCA, along with the presence of an antisense transcript overlapping the 3' exon of SNCA. Moreover, we demonstrate widespread antisense transcription throughout the frontal cortex that is largely not affected by MSA-specific neurodegenerative process. MSA causes a large disruption of lincRNAs in the human brain along with protein coding genes related to iron metabolism and immune response regulation. Most of the lincRNAs specific for MSA were novel. Hence our study uncovers another level of complexity in transcriptional pathology of MSA.

Assigning functions to genes--the main challenge of the post-genomics era.

Genome-sequencing projects yield enormous amounts of information that can lead to revolutions in our understanding of life and provide new platforms for the treatment of human diseases. However, DNA sequencing alone does not provide enough information to determine the molecular pathways of an organism in healthy and disease states. A huge number of gene products await functional characterization. Hence, there is a strong demand for technological solutions that help to assign the functions of proteins and genes. This review discusses high-throughput molecular biology methods, which promise to meet the challenges of the post-genomic era.

High-throughput RNA interference in functional genomics.

RNA interference (RNAi) refers to post-transcriptional silencing of gene expression as a result of the introduction of double-stranded RNA into cells. The application of RNAi in experimental systems has significantly accelerated elucidation of gene functions. In order to facilitate large-scale functional genomics studies using RNAi, several high-throughput approaches have been developed based on microarray or microwell assays. The recent establishment of large libraries of RNAi reagents combined with a variety of detection assays has further improved the performance of functional genome-wide screens in mammalian cells.

High-throughput subcellular protein localization using cell arrays.

Accomplishment of the human and mouse genome projects resulted in accumulation of extensive gene sequence information. However, the information about the biological functions of the identified genes remains a bottleneck of the post-genomic era. Hence, assays providing simple functional information, such as localization of the protein within the cell, can be very helpful in the elucidation of its function. Transfected cell arrays offer a robust platform for protein localization studies. Open reading frames of unknown genes can be linked to a His6-tag or GFP (green fluorescent protein) reporter in expression vectors and subsequently transfected using the cell array. Cellular localization of the transfected proteins is detected either by specific anti-His-tag antibodies or directly by fluorescence of the GFP fusion protein and by counterstaining with organelle-specific dyes. The high throughput of the method in terms of information provided for every single experiment makes this approach superior to classical immunohistological methods for protein localization.

Establishment of T cell-specific and natural killer cell-specific unigene sets: towards high-throughput genomics of leukaemia.

We report the establishment of highly non-redundant unigene sets consisting of cDNA clones derived from T lymphocytes and natural killer cells. Each set consists of 10 506 and 13 409 clones, respectively, arrayed on nylon membranes in duplicate. The sets provide an excellent tool for genome-wide gene expression analysis studies in immunology research.

Analysis of the sequence polymorphism within class II transactivator gene promoters.

The class II transactivator is a major transcriptional factor acting on the promoters of MHC class II genes. Transcription of the CIITA gene is driven by four alternative promoters, which exhibit cell-type-specific activity. The CIITA promoter III (PIII) is constitutively active in B cells, whereas promoter IV (PIV) becomes activated upon interferon-gamma activation. The aim of this study was to investigate whether these two promoters exhibit a sequence variability like the MHC class II promoters do. We isolated PIII and PIV fragments from healthy individuals and rheumatoid arthritis patients and screened them for sequence polymorphisms. Single base pair substitutions within the CIITA PIV were found in 9% of the individuals analyzed. The majority of the substitutions were located upstream of the known cis-acting elements of the promoter. PIII was non-polymorphic. To evaluate the functional relevance of the detected polymorphism we cloned variable PIV upstream of the luciferase reporter gene. Such prepared constructs were transfected into monocytes, melanoma and HeLa cells, which were subsequently stimulated with interferon-gamma. The analysis of promoter activities did not reveal significant differences in all three cell types. We conclude that the level of CIITA expression does not vary within the population. Thus the differences in the level of MHC class II expression, which are observed between individuals, stem for the polymorphisms of the MHC class II promoters themselves.

Expression of the H2-Ea gene is modulated by a polymorphic transcriptional enhancer.

In all vertebrates the major histocompatibility complex (MHC) class II genes are polymorphic in their coding regions as well as in their promoter control elements. This polymorphism correlates with a variability in peptide binding and a variability in transcriptional activities. There is, however, one exception to this rule, which is the mouse H2-Ea gene or the corresponding human DRA gene. So far and for unkown reasons no polymorphism has been observed in these loci. We sequenced the distal transcriptional control elements of the H2-Ea, H2-Eb, and H2-Ab genes from the mouse haplotypes H2d, H2k, H2q, and H2z, and in contrast to the promoter and coding regions a sequence polymorphism can be detected which is limited to the H2-Ea gene. In transfection experiments this polymorphism can be seen to influence haplotype-specifically the transcriptional activities in B cells. This finding strongly suggests an evolutionary pressure towards a haplotype-specific expression pattern in all four MHC class II genes. The genetic differences in control elements of MHC class II genes may well contribute to differential immune reactivities and to immune disorders like allergies or autoimmune diseases.

The distribution of variation in regulatory gene segments, as present in MHC class II promoters.

Diversity in the antigen-binding receptors of the immune system has long been a primary interest of biologists. Recently it has been suggested that polymorphism in regulatory (noncoding) gene segments is of substantial importance as well. Here, we survey the level of variation in MHC class II gene promoters in man and mouse using extensive collections of published sequences together with unpublished sequences recently deposited by us in the EMBL gene bank using the Shannon entropy to quantify diversity. For comparison, we also apply our analysis to distantly related MHC class II promoters, as well as to class I promoters and to class II coding regions. We observe a high level of intraspecies variability, which in mouse but not in man is localized to a significant extent near the binding sites of transcription factors-sites that are conserved over longer evolutionary distances. This localization may both indicate and enhance heterozygote advantage, as the presence of two functionally different promoters would be expected to confer flexibility in the immune response.

Three alternatively spliced variants of the gene coding for the human bone morphogenetic protein-1.

The human bone morphogenetic protein-1 was originally identified as a protein with the capacity to stimulate bone and cartilage growth in vitro. Its gene sequence identified it as an alternatively spliced human homolog of the Drosophila dorsal-ventral patterning tolloid gene and suggested that it activates transforming growth factor-beta-like molecules by proteolytic cleavage. Its expression pattern and its recently identified activity as a procollagen C proteinase, however, suggest that it has a more general function in the early stages of embryogenesis. This view is strengthened by the previous observation of a third alternatively spliced isoform of the gene, called bone morphogenetic protein 1/His. We now show that the gene is expressed in three additional variants, leading to shorter and slightly modified C-termini. The three variants are preferentially expressed in placenta but show individual differences in their expression profiles in other soft tissues.

Polymorphic MHC class II promoters exhibit distinct expression pattern in various antigen-presenting cell lines.

The promoter regions of MHC class II genes are characterized by the presence of conserved sequence motifs called S,X and Y boxes, which are crucial for regulation of transcription of these genes. In humans, promoter polymorphism is known to result in differential transcriptional activity at both inter-locus and inter-allelic levels, but it is not yet known how this relates to tissue-specific expression of MHC class II molecules. We sequenced the 5' regulatory regions of alpha and beta genes of I-A and I-E molecules from four mouse haplotypes and found allelic polymorphisms which were mainly confined to the X box. The promoter sequences of I-Ea genes were non-polymorphic. Transfection of four antigen-presenting cell types with promoter-reporter gene constructs revealed that the promoter sequence polymorphisms result in distinct allele- and tissue-specific activity patterns. Mutagenesis experiments in which the X2 box was reshuffled between I-A beta alleles demonstrated that this box contributes to regulation of differential MHC class II expression in the four cell types. The possibility is discussed that tissue-specific MHC class II expression may control differentiation of T-cell subsets.

Analysis of mRNA expression for interleukin-1 genes on human testicular cells.

We have investigated mRNA expression for IL-1 alpha and IL-1 beta gene on fractionated human testicular cells. Using RT-PCR and Northern blot hybridization technique we detected the presence of IL-1 alpha transcripts, predominantly in the intratubular compartment of the testis, comprising gametogenic and Sertoli cells. We were also able to detect mRNA for IL-1 alpha on the testicular interstitium, but at significantly lower levels. The intertubular compartment of the testis, mainly consisting of macrophages and Leydig cells, appeared however, to be a site for IL-1 beta gene expression. Our experimental data confirm previous results obtained in animal models indicating that the testis is capable of producing interleukin-1 under physiological conditions. Testicular IL-1 may function as a tissue-specific factor modulating both spermato- and steroidogenic activity of human testis.

Analysis of mRNA for class I HLA on human gametogenic cells.

We have studied mRNA expression for Class I HLA (human leukocyte antigen) on male germ cells by amplification of gene fragments in PCR technique and by Northern hybridization. RNA was extracted from fractionated gametogenic cells (isolated from testis) and reversely transcribed. Then, cDNA was amplified for specific HLA sequence (HLA, -A, -B, -C). The specificity of this product was confirmed in "nested" PCR of 400 bp gene fragment coding for alpha 2 domain, alpha 3 domain, and the transmembrane portion of Class I HLA. The results indicate minimal expression of classical Class I HLA on gametogenic cells. Northern hybridization with 669 bp cDNA fragment (spanning for alpha 3 domain, transmembrane, cytoplasmic, and 3' untranslated region) resulted in a low intensity signal from gametogenic cell fractions and confirmed our findings obtained by PCR. The minimal expression of classical HLA antigens may create a neutral cover for the male reproductive system, thereby preventing an immunological response during germ cell differentiation.

In situ localization of HLA class I mRNA in human testis.

We have used a 0.35-kilobase (kb) antisense RNA probe complementary to the monomorphic regions of both classical and nonclassical HLA class I sequences to detect histocompatibility-class-I-antigen-specific mRNA in human testicular tissue. The message has been clearly detected in the interstitium while less intensive staining was revealed in the peribasal compartment of the seminiferous epithelium.