Categories // Cell development biology //
There’s nothing crabby about RNA sequencing
10 DEC 2019CELL DEVELOPMENT BIOLOGY
Researchers have utilized the neural networks of crabs to validate the use of RNA sequencing for the identification of single neurons.
A team of researchers, led by David Schulz (University of Missouri, MO, USA), have demonstrated the ‘clawsome’ power of RNA sequencing for the anatomical and functional identification of single neurons.
The use of RNA sequencing is widespread for the identification of cells in the nervous system. However, previous classification studies have been impeded by other features of the cells. This work aimed to validate the accuracy of single-cell transcriptional profiling for the identification of individual neurons.
Speaking to BioTechniques, Schulz commented, “RNA sequencing is, and will be, used not only to understand how neurons work under typical conditions – and what makes them distinct from one another – but also to shed light on how development, growth, learning, injury, and disease change neurons over the lifetime of an individual.”
RNA sequencing and reverse transcriptase PCR were performed on two ganglia from the crab Cancer borealis, a model in which the identified neurons had previously been classified. The team essentially worked backward from previous results to validate the use of RNA sequencing.
Schulz explained, “with known cell identity to work from, we could blind ourselves to their identities for some of the analyses and see how well unbiased statistical methods perform in terms of recapitulating known biologically/physiologically known cell identity.”
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Each neuron analyzed exhibited distinct gene expression patterns; however, expression profiles were not enough to accurately identify the neurons. Unbiased clustering analysis and supervised machine learning were also integrated into this approach.
The results demonstrated that transcriptional profiling for the assignment of neuronal identity is most accurate when combined with information regarding the physiology and morphology of the cells.
Identifying and understanding the operation of a single neuron has implications for developing targeted therapies for neurodegenerative diseases. RNA sequencing has the potential to inform our knowledge of how neurons are affected by injury and disease.
Understanding the intersect between variability, environment and genetics could advance insight into how individuals are differently affected by neurological diseases, contributing to the precision medicine revolution.
The team now hopes to utilize the knowledge gained from this study to determine how neurons are affected in spinal cord injuries.
“We are working to understand how injuries and diseases that cause changes to the normal activity of neural networks affect the neuronal gene expression profiles, and what this can tell us about how even indirect effects can contribute to pathology “downstream” of the actual insult,” concluded Schulz.
If you were interested in this post and would like to learn more, check out the Cells as Models track of our Online Event: Advancing Precision Medicine 2019 and register for free today. To get more information visit the event page here.
WRITTEN BYCaitlin Killen
SOURCENorthcutt AJ, Kick DR, Otopalik AG et al. Molecular profiling of single neurons of known identity in two ganglia from the crab Cancer borealis. Proc. Natl. Acad. Sci. USA. doi: 10.1073/pnas.1911413116 (2019);https://www.pnas.org/content/early/2019/12/04/1911413116https://news.missouri.edu/2019/think-like-a-crab/
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© 2019 BIOTECHNIQUES
Adenine forms bonds with uracil, and guanine forms bonds with cytosine. In this way, we say that adenine is complementary to uracil and that guanine is complementary to cytosine. The first three bases are also found in DNA, but uracil replaces thymine as a complement to adenine.
RNA also contains ribose as opposed to deoxyribose found in DNA. These differences result in RNA being chemically more reactive than DNA. This makes it the more suitable molecule to take part in cell reactions.
Protein synthesis RNAs[change | change source]
Messenger RNA[change | change source]
This is done by messenger RNA (mRNA). A single strand of DNA is the blueprint for the mRNA which is transcribed from that DNA strand. The sequence of base pairs is transcribed from DNA by an enzyme called RNA polymerase. Then the mRNA moves from the nucleus to the ribosomes in the cytoplasm to form proteins. The mRNA translates the sequence of base pairs into a sequence of amino acids to form proteins. This process is called translation.
DNA does not leave the nucleus for various reasons. DNA is a very long molecule, and is bound in with proteins, called histones, in the chromosomes. mRNA, on the other hand is able to move and to react with various cell enzymes. Once transcribed, the mRNA leaves the nucleus and moves to the ribosomes.
Two kinds of non-coding RNAs help in the process of building proteins in the cell. They are transfer RNA (tRNA) and ribosomal RNA (rRNA).
tRNA[change | change source]
Transfer RNA (tRNA) is a short molecule of about 80 nucleotides which carries a specific amino acid to the polypeptide chain at a ribosome. There is a different tRNA for each amino acid. Each one has a site for the amino acid to attach, and an anti-codon to match the codon on the mRNA. For example, codons UUU or UUC code for the amino acid phenylalanine.
rRNA[change | change source]
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. rRNA is extremely abundant and makes up 80% of the 10 mg/ml RNA found in a typical eukaryotic cytoplasm.
snRNAs[change | change source]
Small nuclear RNAs (snRNA) join with proteins to form spliceosomes. The spliceosomes govern alternative splicing. Genes code for proteins in bits called exons. The bits can be joined together in different ways to make different mRNAs. Thus, from one gene many proteins can be made. This is the process of alternative splicing. Any unwanted versions of the protein get chopped up by proteases, and the chemical bits re-used.
Regulatory RNAs[change | change source]
There are a number of RNAs which regulate genes, that is, they regulate the rate at which genes are transcribed or translated.
miRNA[change | change source]
siRNA[change | change source]
Small interfering RNAs (sometimes called silencing RNAs) interfere with the expression of a specific gene. They are quite small (20/25 nucleotides) double-stranded molecules. Their discovery has caused a surge in biomedical research and drug development.
Parasitic and other RNAs[change | change source]
Retrotransposons[change | change source]
Transposons are only one of several types of mobile genetic elements. Retrotransposons copy themselves in two stages: first from DNA to RNA by transcription, then from RNA back to DNA by reverse transcription. The DNA copy is then inserted into the genome in a new position. Retrotransposons behave very similarly to retroviruses, such as HIV.
Viral genomes[change | change source]
Viral genomes, which are usually RNA, take over the cell machinery and make both new viral RNA and the protein coat of the virus.
Phage genomes[change | change source]
Phage genomes are quite varied. The genetic material can be ssRNA (single-stranded RNA), dsRNA (double-stranded RNA), ssDNA (single-stranded DNA), or dsDNA (double-stranded DNA). It may be between 5 and 500 kilo base pairs long with either circular or linear arrangement. Bacteriophages are usually between 20 and 200 nanometers in size.
References[change | change source]
- Only the most important are described here. A complete list is available at en:List of RNAs.
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|chapterurl=missing title (help). RNA and the regulation of gene expression: a hidden layer of complexity. Caister Academic Press. ISBN 978-1-904455-25-7.
- The Nobel Prize in Physiology or Medicine 2006. RNA Interference
- Lee R.C. & Ambros V. 2001. An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862-864.
- Lau N.C. et al 2001. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858-862.
- Hamilton A. & Baulcombe D (1999). “A species of small antisense RNA in posttranscriptional gene silencing in plants”. Science. 286: 950–2. doi:10.1126/science.286.5441.950. PMID 10542148. First description of siRNAs.
- Hannon G. & Rossi J (2004). “Unlocking the potential of the human genome with RNA interference”. Nature. 431: 371–8. doi:10.1038/nature02870. PMID 15372045.
- Bacteriophage MS2