转录组的现状及未来

In the last decades, transcriptome profiling has been one of the most utilized approaches to investigate human diseases at the molecular level. Through expression studies, many molecular biomarkers and therapeutic targets have been found for several human pathologies. This number is continuously increasing thanks to total RNA sequencing. Indeed, this new technology has completely revolutionized transcriptome analysis allowing the quantification of gene expression levels and allele-specific expression in a single experiment, as well as to identify novel genes, splice isoforms, fusion transcripts, and to investigate the world of non-coding RNA at an unprecedented level. RNA sequencing has also been employed in important projects, like ENCODE (Encyclopedia of the regulatory elements) and TCGA (The Cancer Genome Atlas), to provide a snapshot of the transcriptome of dozens of cell lines and thousands of primary tumor specimens. Moreover, these studies have also paved the way to the development of data integration approaches in order to facilitate management and analysis of data and to identify novel disease markers and molecular targets to use in the clinics. In this scenario, several ongoing clinical trials utilize transcriptome profiling throughRNAsequencing strategies as an important instrument in the diagnosis of numerous human pathologies.

第一页PPT:转录组的发展，从以前到现在

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1. The first approach to profiling human transcriptomes started with the publication of a database with human ESTs (expressed sequence tags), short sequences of cDNA clones obtained by the first DNA sequencers.
2. SAGE (serial analysis of gene expression) and microarray, employing complementary probe hybridization, tried to quantify gene expression on a global basis,这也是转录组发展最快的时刻，很多重要基因及通路的发现都是在这个策略中找到的
3. Contextually, the quantitative reverse transcription PCR (qRT-PCR) method was often applied to validate the results from high-throughput platforms.Indeed, this technique is considered the gold standard system for measuring transcript levels since it is fast, reliable, reproducible, sensitive and accurate, even though it is able to analyze one or a few genes in a single assay and could provide incomplete or misleading data if alternative splicing isoforms are present; in these cases proper primer design becomes a very critical step in the validation procedure.随着发展也就有了dPCR这种微定量方法
4. RNA-seq

第二页PPT:二代测序给转录组研究带来了新视角

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1. An important advantage of this technology is the possibility of also detecting and quantifying An important advantage of this technology is the possibility of also detecting and quantifying low-expressed genes that could not be revealed by microarray analysis
2. more information on post-transcriptional RNA editing, especially splicing
3. 一个详尽的例子：A relationship between defective alternative splicing and human pathologies was already demonstrated through sequencing analysis many years ago, when our group also discovered a new exon in the RPGR retinitis pigmentosa GTPase regulator) gene, preferentially expressed in mouse and bovine retina, which was mutated in atients with X-linked Retinitis pigmentosa （Nat Genet. 2000 Aug;25(4):462-6.）
4. Schematic representation of the structure of RPGR (top) and alternative transcripts. Besides the 19 exons reported previously1, we documented 5 additional exons: 15b1, 15b2, 15a, ORF14 and ORF15. Exons 15b1 and 15b2 are two overlapping exons within intron 15, which use alternative acceptor splice sites and the same donor site. Their inclusion is predicted to result in premature termination of translation. Exon 15a, which is a third internal exon within intron 15 and also encodes a premature stop codon, is identical with the exon 15a reported previously11. Exon ORF14 corresponds to the mouse exon 14-14a-15 (ref. 11); its inclusion does not disrupt the reading frame. Exon ORF15 is a large 3´ terminal exon consisting of exon 15 and extending into part of intron 15. An exon containing reported mutations in XLRP is shown as a black box, a poly(A) tract as a bold line, a predicted stop codon as an asterisk. b–i, The results of RT–PCR analysis of total RNA from human tissues and cell lines. The regions analysed are represented schematically on the right. Relatively abundant transcripts were amplified without the use of exon-specific or nested primers. Besides the 19-exon mRNA reported previously1, 2 such transcripts were found in retina: one was widely expressed and lacked exons 14 and 15 (h), another was preferentially expressed in the retina and contained exon 15a

第三页PPT:除了对于mRNA的帮助以外，还是我们看到了更多其他的RNA研究

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Messenger RNA (mRNA) is the RNA that carries information from DNA to the ribosome, the sites of protein synthesis (translation "Translation (biology)")) in the cell. The coding sequence of the mRNA determines the amino acidsequence in the protein that is produced.[27] However, many RNAs do not code for protein (about 97% of the transcriptional output is non-protein-coding in eukaryotes[28][29][30][31]).

These so-called non-coding RNAs ("ncRNA") can be encoded by their own genes (RNA genes), but can also derive from mRNA introns.[32] The most prominent examples of non-coding RNAs are transfer RNA(tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation.[4] There are also non-coding RNAs involved in gene regulation, RNA processing and other roles. Certain RNAs are able to catalyse chemical reactions such as cutting and ligating other RNA molecules,[33] and the catalysis of peptide bond formation in the ribosome;[7] these are known as ribozymes.

According to the length of RNA chain, RNA includes small RNA and long RNA.[34] Usually, small RNAs are shorter than 200 nt in length, and long RNAs are greater than 200 nt long.[35] Long RNAs, also called large RNAs, mainly include long non-coding RNA (lncRNA) and mRNA. Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA)[36] and small rDNA-derived RNA (srRNA).[37]

Messenger RNA (mRNA) carries information about a protein sequence to the ribosomes, the protein synthesis factories in the cell. It is coded so that every three nucleotides (a codon) corresponds to one amino acid. In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it is processed to mature mRNA. This removes its introns—non-coding sections of the pre-mRNA. The mRNA is then exported from the nucleus to the cytoplasm, where it is bound to ribosomes and translated "Translation (biology)") into its corresponding protein form with the help of tRNA. In prokaryotic cells, which do not have nucleus and cytoplasm compartments, mRNA can bind to ribosomes while it is being transcribed from DNA. After a certain amount of time, the message degrades into its component nucleotides with the assistance of ribonucleases.[27]

Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino acid attachment and an anticodon region for codon recognition that binds to a specific sequence on the messenger RNA chain through hydrogen bonding.[32]

Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. Three of the rRNA molecules are synthesized in the nucleolus, and one is synthesized elsewhere. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes may be attached to a single mRNA at any time.[27] Nearly all the RNA found in a typical eukaryotic cell is rRNA.

Transfer-messenger RNA (tmRNA) is found in many bacteria and plastids. It tags proteins encoded by mRNAs that lack stop codons for degradation and prevents the ribosome from stalling.[38]

Several types of RNA can downregulate gene expression by being complementary to a part of an mRNA or a gene's DNA.[39][40] MicroRNAs (miRNA; 21-22 nt) are found in eukaryotes and act through RNA interference (RNAi), where an effector complex of miRNA and enzymes can cleave complementary mRNA, block the mRNA from being translated, or accelerate its degradation.[41][42]

While small interfering RNAs (siRNA; 20-25 nt) are often produced by breakdown of viral RNA, there are also endogenous sources of siRNAs.[43][44] siRNAs act through RNA interference in a fashion similar to miRNAs. Some miRNAs and siRNAs can cause genes they target to be methylated, thereby decreasing or increasing transcription of those genes.[45][46][47] Animals have Piwi-interacting RNAs (piRNA; 29-30 nt) that are active in germline cells and are thought to be a defense against transposons and play a role in gametogenesis.[48][49]

Many prokaryotes have CRISPR RNAs, a regulatory system similar to RNA interference.[50] Antisense RNAs are widespread; most downregulate a gene, but a few are activators of transcription.[51] One way antisense RNA can act is by binding to an mRNA, forming double-stranded RNA that is enzymatically degraded.[52] There are many long noncoding RNAs that regulate genes in eukaryotes,[53] one such RNA is Xist "XIST (gene)"), which coats one X chromosome in female mammals and inactivates it.[54]

An mRNA may contain regulatory elements itself, such as riboswitches, in the 5' untranslated region or 3' untranslated region; these cis-regulatory elements regulate the activity of that mRNA.[55] The untranslated regions can also contain elements that regulate other genes.[56]

Uridine to pseudouridine is a common RNA modification.

Many RNAs are involved in modifying other RNAs. Introns are spliced "Splicing (genetics)") out of pre-mRNA by spliceosomes, which contain several small nuclear RNAs (snRNA),[4] or the introns can be ribozymes that are spliced by themselves.[57] RNA can also be altered by having its nucleotides modified to nucleotides other than A, C, G and U. In eukaryotes, modifications of RNA nucleotides are in general directed by small nucleolar RNAs(snoRNA; 60–300 nt),[32] found in the nucleolus and cajal bodies. snoRNAs associate with enzymes and guide them to a spot on an RNA by basepairing to that RNA. These enzymes then perform the nucleotide modification. rRNAs and tRNAs are extensively modified, but snRNAs and mRNAs can also be the target of base modification.[58][59] RNA can also be methylated.[60][61]

Like DNA, RNA can carry genetic information. RNA viruses have genomes composed of RNA that encodes a number of proteins. The viral genome is replicated by some of those proteins, while other proteins protect the genome as the virus particle moves to a new host cell. Viroids are another group of pathogens, but they consist only of RNA, do not encode any protein and are replicated by a host plant cell's polymerase.[62]

Reverse transcribing viruses replicate their genomes by reverse transcribing DNA copies from their RNA; these DNA copies are then transcribed to new RNA. Retrotransposons also spread by copying DNA and RNA from one another,[63] and telomerase contains an RNA that is used as template for building the ends of eukaryotic chromosomes.[64]

Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA found in all cells. dsRNA forms the genetic material of some viruses (double-stranded RNA viruses). Double-stranded RNA such as viral RNA or siRNA can trigger RNA interference in eukaryotes, as well as interferon response in vertebrates.[65][66][67][68]

In the late 1970s, it was shown that there is a single stranded covalently closed, i.e. circular form of RNA expressed throughout the animal and plant kingdom (see circRNA).[69] circRNAs are thought to arise via a "back-splice" reaction where the spliceosome joins a downstream donor to an upstream acceptor splice site. So far the function of circRNAs is largely unknown, although for few examples a microRNA sponging activity has been demonstrated.

第四页PPT:Transcriptome Studies by NGS: theWork in Progress

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For instance, a very recent study using the unbiased single-cell RNA-Seq of ~2400 cells has revealed new types of human blood dendritic cells, monocytes and circulating progenitors, thus providing a revised taxonomy, which will support immune monitoring in both health and disease （Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science 2017, 356, eaah4573.）

第五页PPT:未来，多组学（至少和表观遗传联系）（Biochemical Journal (2017) 474 885–896 DOI: 10.1042/BCJ20161047）

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The epigenetic modifications and alternative splicing are individually known to be altered in cancer cells thus it is possible that aberrant epigenetic changes may lead to the generation of cancer-specific splice isoforms and promote tumorigenesis

参考文献

• Transcriptome Profiling in Human Diseases: New Advances and Perspectives
• RNA editing-dependent epitranscriptome diversity in cancer stem cells
• A survey of best practices for RNA-seq data analysis