RNAtrain:Scientific Background
Non-protein-coding RNA
What is non-coding RNA?
For quite a long time, it was assumed that RNA molecules were merely the ephemeral intermediates between DNA and proteins. Indeed, messenger RNA (mRNA) is copied from genes and later translated by ribosomes into peptides, with the help of transfer RNA (tRNA), obediently bringing amino acids to the protein building site, leading to the "one gene, one protein" hypothesis.
Over the last five decades however, it has become more and more clear, that RNA molecules have a quite exciting life on their own and that there are tons of different types of RNA molecules in one single cell. Far from being simple messengers, RNA can... (Written by Sophia Häfner)
Autophagy
What is autophagy good for?
If you have not eaten the whole day and you are getting very hungry, your body starts to use its sugar and fat reserves in order to maintain sufficient nutrient supply for body functions. Maintaining a nutrient balance is not only important in whole organisms, but also on the cellular level. In times of insufficient nutrient supply, cells turn on a recycling machinery called autophagy (auto = self, phagy = eating). In a simple picture, the cells form rather big vesicles in their cytoplasm that are called autophagosomes. In some way, these autophagosomes resemble “trash bags” in which the cell collects old protein aggregates, damaged organelles, other cellular debris and even bacteria. In order to disrupt the “trash cargo”, the cell adds a “chemical cocktail” (certain enzymes in an acidic milieu) provided by another vesicle, called the lysosome. After fusion of the autophagosome with the acidic lysosome the “trash cargo” becomes broken down into its components (small peptides, amino acids etc). The recycling circle closes when the components are restored to the cell and thus available for the production of new cell material. Interestingly, this recycling system is not only active during times of starvation. Even under nutrient-rich conditions, there is a basal but lower level of autophagy going on, in order to prevent accumulation of damaged or aged proteins and mitochondria. Such an accumulation can cause severe dysfunction of the cells and it has been associated with a range of human diseases affecting the nervous system, the immune system and cancer. Given the importance of autophagy, it needs tight regulation in order to respond fast and accurate to environmental changes. There are not only many autophagy-specific key proteins known that can up- or down-regulate autophagy, but also some RNA molecules. We know e.g. that small non-coding RNAs (e.g. microRNAs) can block proteins, which would usually turn on autophagy during starvation. We are now interested to investigate whether there are also long non-coding RNAs that can regulate this essential cellular recycling machinery. (Written by Imke Ulken)
Senescence
Any living entity is born, grows, and eventually dies. The process of becoming old is called “senescence”, a word referred either to the aging of the whole organism or to cellular senescence. Because cells are the fundamental building blocks of our body, it is commonly believed that cellular senescence underlies organismal senescence. A cell is defined senescent when it is no longer capable of dividing but still alive and metabolically active. When cells become senescent they change their morphology (enlarge and elongated shape), DNA organization and protein secrection compared to proliferative cells. Senescence happens when cells have reached their maximum lifespan: this is called “replicative senescence” and is mainly due to the telomeres shortening. Telomeres are small segments of DNA at each end of the chromosomes and ensure DNA stability. When cells duplicate their genome, telomeres cannot be entirely copied along with the rest of the DNA, so they get shorter with each new cell division until they shorten to a critical length (Hayflick limit). However, cells can become senescent prematurely due to different stresses such as DNA damage and activation of genes that can promote cancer (oncogenes). This last type of senescence is called “oncogene-induced senescence” (OIS). Cancer is defined as a large group of diseases that involve abnormal cell growth with the potential to invade other parts of the body. As senescence leads to an irreversible growth arrest, it is considered a barrier against cancer development.
Besides a large number of proteins, also non-coding RNAs molecules (RNAs that do not encode for proteins) can regulate cellular senescence. Our efforts are directed toward the identification of long non-coding RNAs (non-coding RNAs longer than 200 nucleotides) that play a role in OIS and so can be important in preventing cancer development. (Written by Giulia Maglieri, Elena Grossi, Li Li)
Cancer/Neuroblastoma
Cancer is a very complex disease that originates because of uncontrolled cellular events. Cancer cells gain some unfavorable features where they divide indefinitely and don’t respond to programmable cellular death signals; giving rise to different types of tumors. Among different types of cancers, neuroblastoma (NB) is the one that arises from improper development and differentiation of neural crest cells. It is considered as the third most common tumor affecting infants and comprises around 7% of the total tumors observed in children. Neuroblastoma contains many molecular subtypes; including low-risk and high-risk tumors. The low-risk tumors are not associated with metastasis, rather they tend to differentiate into mature cells and respond to chemotherapy. On the other hand, high-risk tumors usually spread into different parts of the body and they show unfavorable clinical outcome due to their aggressive nature.
Neuroblastoma is characterized with several genomic alterations; such as focal amplification (1) of the chromosomal segment 2p24. MYCN is a well-studied oncogene which maps to the amplified region. Genetic amplification of MYCN gene is observed in 25-30% of NBs and is mostly associated with aggressive high-risk tumors. In addition, many long non-coding RNAs (lncRNAs) are co-amplified with MYCN gene and they show a similar pattern of expression.
Recently, the neuroblastoma associated transcript 1 lncRNA (NBAT1) was reported as a risk factor which is differentially expressed between high-risk and low-risk NBs. Less expression of NBAT1 leads to more proliferation of cancer cells, while higher expression level induces cellular differentiation and maturation. Using NBAT1 expression as an independent prognostic marker enables the prediction of overall survival and clinical outcome among different groups.
Systematic investigation of stage-specific as well as subtype-specific lncRNAs will lead to a more comprehensive understanding of NB progression. Also, it may help in revealing more about regulatory networks that govern neuronal differentiation and proliferation. Modulating the expression of important lncRNAs might provide a possible treatment for NB patients. (Written by Mohamad Gendy)
Myogenesis
Skeletal muscle is made up of individual components known as myofibers, which are multinucleated cells formed during muscle development in a process termed myogenesis. While a mammalian embryo is developing, progenitor cells (1) become determined for the myogenic lineage (determination step) and give rise to proliferating myoblasts. Myoblasts will transform to myocytes which withdraw from the cell cycle, and fuse to one other to form multinucleated myotubes (see figure). Myotubes at their final stage of differentiation will give rise to myofibers. During adult muscle regeneration and growth, myoblasts are derived from resident muscle precursor cells (3), termed satellite cells. Satellite cells are mitotically quiescent (2), and they reside in muscle tissues in association with myofibers. Upon a growth stimulus or injury, satellite cells activate, proliferate and undergo differentiation process to produce new myofibers. Myogenesis is a highly organized process, many transcription factors (4) are known to be important for regulation of different steps of this procedure (see figure). miRNAs (5) and lncRNAs are another factors which recently have been discovered as myogenesis regulators. (Written by Sama Shamloo)
Glossery: (1) A progenitor cell is a biological cell that, like a stem cell, has a tendency to differentiate into a specific type of cell, but is already more specific than a stem cell and is pushed to differentiate into its "target" cell.) (2) Quiescent cell is neither dividing nor preparing to divide. (3) Precursor cells are stem cells that have developed to the stage where they are committed to forming a particular type of cell. (4) Transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to RNA. (5) A microRNA is a small non-coding RNA molecule found in plants, animals, and some viruses, which functions in RNA silencing and post-transcriptional regulation of gene expression.
Oocyte to Embryo Transition
Oocyte to zygote transition (OZT) is the most important transitions in life. OZT refers to transformation of a differentiated oocyte into a zygote, which has potential to develop into a new organism – i.e. zygotic cells acquire totipotency, which refers to their ability to give rise to any embryonic and extra embryonic cell types. Oocytes start to form already during embryonic development when oocyte precursors (primordial germ cells) migrate into the future gonad. In mice and humans, oocytes are already formed at birth and rest in the ovary. Some of the oocytes subsequently enter growth phase in response to periodical hormonal stimulation in sexually mature females. During the growth phase, oocytes grow in size and their genes synthesize factors, which will support future development. Factors stored include a wide range of RNA molecules. Fully-grown oocytes stop their transcription and are released from the ovary for fertilization. Upon fertilization, a massive reprogramming of gene expression takes place, which transforms a differentiated oocyte into the totipotent zygote. This reprogramming is driven by maternal factors stored in the oocyte until the control is taken over by zygotic gene expression. Therefore both maternal and zygotic factors contribute to this complex transition. In mice, the major phase of zygotic genome activation takes place at the 2-cell stage while in other mammals usually occurs a bit later (4-16-cell stages). Although several studies have been performed to unveil the mystery of OZT, key factors involved are yet to be identified. Our lab studies contribution of non-coding RNA to the genome reprogramming during OZT in mice. (Written by Sravya Ganesh)
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Colorectal Cancer
If you daily consume high intake of fat, alcohol, red meat, smoke and lack of physical exercise, you may belong to a group of high risk of colorectal cancer (CRC). CRC originates from the epithelial cells lining in the colon or rectum, some of which have abnormal growth activity and ability to invade or spread to other parts of the body. Currently, scientists are putting high effort to find the genes or signaling pathways which lead normal cells to become cancerous with the hope to early prevent the tumor development and metastasis. Genome-scale analysis and next generation sequencing has revealed that many protein-coding genes and non-protein-coding genes are all involved in causing abnormal colorectal cancer cells.
These abnormal cells are most frequently as a result of mutations, which can be inherited or acquired, in the Wnt-signaling pathway. APC gene is the most commonly mutated gene in all colorectal cancer. The APC gene produces APC protein, which prevents the accumulation of β-catenin protein. When β-catenin accumulates, they translocates (moves) into the nucleus, binds to DNA, and activates the transcription of proto-oncogenes. Other mutations occur in cancer-related genes, such as p53 or BAX.
With the advantages of next generation sequencing (refer to RNA-seq from Foivos Gypas), there are more and more evidence indicating that non-coding RNAs, either small non-coding RNAs or long non-coding RNAs, are frequently aberrantly expressed in cancers, and some of them have been implicated in CRC development and metastasis. Further characterization of these non-coding RNAs will not only give us new insights into the molecular biology of CRC development but also provide us potential non-coding RNA therapeutic prevention in the future. (Written by Hung Ho)
Development of siRNA pools
Due to the rapidly expanding role of RNA in human health and disease, scientists are developing methods to specifically regulate different RNAs to understand their function. siRNA or silencing RNA was discovered in the 1990s by Craig Mello and Andrew Fire, for which they were awarded the Nobel Prize in Physiology/Medicine in 2006. They found that introducing double-stranded RNA into worms (C. Elegans) reduced the expression of certain genes. It was later revealed that on entering the cell, double-stranded RNA is cleaved into smaller lengths of ~21-25 ribonucleotides by an enzyme called Dicer. These small intermediates carry out the gene knockdown effect, specifically the antisense strand would bind to a complementary RNA sequence from an endogenous target gene, recruiting it to a protein complex called RISC or RNA-induced silencing complex. This complex contains Argonaute proteins which then carry out the “slicing” of the target gene, hence silencing its expression. This heralded a burst in siRNA research as it provided a simple and fast way to regulate gene expression. However, it slowly became clear that these siRNAs had various off-target effects (i.e. that one siRNA can bind specidically to several mRNAs, resulting in lower specificity of the knockdown). siPOOLs were therefore developed by siTOOLs Biotech GmbH. siPOOLs are complex pools of accurately defined siRNAs and dramatically reduce off-target effects while producing robust target gene knock-down. They therefore deliver clean and reliable phenotypic data. My work focusses on improving the development of siPOOLs and developing siPOOLs against non-coding RNA. (Written by Catherine Goh)
RNA-seq
RNA-seq or RNA sequencing is a recently developed technology based on next-generation sequencing that is rapidly replacing the older microarray technology. RNA-seq has a broad range of applications, but one of the most important is to detect and quantify specific RNAs (coding or non coding) at a given moment in time. An RNA-seq experiment can be devided in two parts. The first part is the experimental, where samples are prepared and sequenced and the second part is the computational where data are analyzed. In order to perform an RNA-seq experiment (see figure below), the first thing one should do is to extract the RNA samples. The samples are then cut into small pieces (RNA fragments) and reverse transcribed and amplified in order to generate multiple cDNA fragments. Then these fragments are inserted into a next generation sequencer that generates reads. Reads are the sequences of the small fragments. After this step data are analyzed computationally, since the reads is actually a big text file. In order to find which transcripts or genes are expressed you need to align the reads to the genome (or the transcriptome). A good program to align reads is segemehl. Aligning reads is a computationally expensive task, so your computer should be supplied with sufficient amount of memory, processing power and disk space. You can see the results of the alignment using a genome browser. A nice solution is IGV. Apart from viewing results you can also quantify, the expression level of genes or transcripts. This can be done either by counting how many reads map to a specific location, or by using some more advanced probabilistic methods. Some of these methods can be found here.
(Written by Foivos Gypas)
