[글쓴이:] naturecoding

Regulator of mRNA, miRNA and siRNA

The amount of mRNA is determined by the balance between its transcription rate from DNA and its degradation rate by RNases.

mRNA and qPCR: Understanding Gene Expression (3)

Does the quantity of mRNA perfectly correspond to the amount of protein? Not exactly. The translation of mRNA into protein can be inhibited by very short RNA molecules, such as microRNA (miRNA) and small interfering RNA (siRNA). These regulatory RNAs bind to target mRNA and suppress its translation or promote its degradation, thereby influencing protein production.

What are the differences between miRNA and siRNA? Their mechanisms are quite similar. Both involve specific RNA molecules binding to mRNA, preventing RNA transcriptase from functioning properly. For example, imagine a railway track designed for train wheels; if the track becomes too thick in certain areas, the train will derail instead of following the path smoothly.

miRNA is primarily endogenous, meaning it is naturally produced within cells. It does not perfectly match the target mRNA but binds with slight mismatches in the base sequence. Additionally, miRNA has a broader range of targets, acting somewhat like a grenade, where its inhibitory strength can vary depending on the type of RNA it binds to. On the other hand, siRNA matches the target mRNA perfectly. While it can be naturally produced within cells, it is most commonly synthesized in laboratories and introduced into cells to inhibit specific genes for experimental purposes. Unlike miRNA, siRNA functions like a sniper rifle, precisely targeting and silencing a single gene with high specificity.

Research of miRNA for Diagnosis

This miRNA was a significant topic that won the Nobel Prize in 2024, highlighting its profound importance. However, studying miRNA manually, gene by gene, is not feasible for human eyes and hands alone. Therefore, this field heavily relies on computational programs for research. The ability to process big data is crucial in this context, and it plays a pivotal role in bioinformatics, driving advancements in the field of life sciences. One example is the critical role miRNA plays in the development of brain cells. By measuring the concentration of specific miRNAs, it is possible to assess the normality of a fetus’s brain. For instance, miR-210 and miR-374a are notable examples. These two miRNAs are particularly useful in evaluating the hypoxic conditions in a fetus’s brain, providing valuable insights into fetal brain health.

https://pmc.ncbi.nlm.nih.gov/articles/PMC5853646

Limitation of miRNA and it’s future

If we have a precise map of miRNAs, we could identify which miRNAs are deficient in individuals and supplement them accordingly or determine which miRNAs are effective for specific diseases. This could enable disease treatments that are far more specific than chemical-based drugs. Indeed, there have been various attempts to develop drugs using this concept. However, as previously mentioned, miRNAs function like “grenades,” leading to unexpected toxicity in many cases.

On the other hand, siRNA-based approaches, which operate more like “sniper rifles,” have seen greater success, with about six siRNA-based drugs approved by the FDA. In contrast, miRNA-based therapies have been progressing slowly. Challenges remain in delivering miRNAs to precise target cells, managing unexpected cellular toxicity, and evaluating their stability.

https://www.nature.com/articles/s41587-024-02480-0

According to a paper published in November 2024, some miRNA-based drugs have reached phase 1 or phase 2 clinical trials, but many fail toxicity tests. Recently, the focus has shifted more toward diagnostics. It seems miRNA is being prioritized for diagnostic applications, while siRNA is advancing in the therapeutic field. Understanding miRNA as a tool for diagnosis and siRNA as the therapeutic counterpart provides a clear picture of current trends.

mRNA’s importance

mRNA (messenger RNA) plays a crucial role in the flow of genetic information within a cell. mRNA acts as a messenger that carries genetic instructions from the DNA in the cell’s nucleus to the ribosomes in the cytoplasm, where proteins are synthesized. From the central dogma, which describes genetic information from DNA to RNA and then to proteins, we have learned the importance of RNA in last story.

Watson, Crick, and Franklin’s DNA Story: Central dogma and RNA (2)

Let’s delve deeper into RNA itself. Until now, three types of RNA have been considered crucial: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). Among these, mRNA contains the direct instructions for coding proteins. Therefore, textbooks primarily focus on these three types of RNA. Most people are familiar with the fact that mRNA uses codons—three-base sequences—between the start codon and stop codon to specify individual amino acids. Beyond this, extensive research has been conducted on mRNA, including mechanisms like the poly-A tail, which protects mRNA, and the 5′ cap, where RNA synthesis begins. Here, mRNA is an incredibly useful molecule in biology. By measuring the concentration of mRNA in a cell, we can infer the amount of protein present, as mRNA is absolutely essential for protein synthesis. mRNA analysis is typically performed using qPCR (quantitative PCR), which allows for precise measurement of mRNA levels in cells.

What is coding sequence (CDS)

The region of RNA that encodes the information for a protein, spanning from the start codon to the stop codon, is referred to as the Coding Sequence (CDS). Although RNA molecules that code for a single protein may vary in other regions (e.g., untranslated regions or poly-A tails), they ultimately produce the same protein if their CDS is identical. Therefore, when performing qPCR, the CDS should be the primary target to ensure the accurate quantification of the RNA related to the specific protein.

https://www.ncbi.nlm.nih.gov

On the site, you input nucleotide and then specify the protein of interest.

Click the gene name and select the species (Homo sapiens).

After select mRNA sequence (H.Sapiens mRNA for albumin)

Click CDS (Coding Sequence)

You can locate the CDS sequence by identifying the ATG start codon and the TAA stop codon. The region between these two codons is the CDS (Coding Sequence). Any part of the sequence outside of these codons is considered the non-CDS part, such as untranslated regions (UTRs). If you want to find primer, you can use CDS region to Primer blast.

https://www.ncbi.nlm.nih.gov/tools/primer-blast

After copying the CDS sequence, paste it into the yellow section of the Primer BLAST tool. Adjust the other parameters accordingly. Generally, you prefer a PCR product size of about 90-150 bp, a Tm of 60°C, and a GC content between 45-55%. This will help you design primers that work well for your experiment.

Once the results are generated, you should select primers that show only a single mRNA for a specific protein. If more than one result appears, it means the result is based on multiple mRNA sequences, which reduces the reliability. Afterward, you need to check for self-loops and assess how well the two primers bind to each other. This will help ensure that the primers are designed correctly for your PCR experiment.

mRNA is a single-stranded molecule and is highly prone to degradation. Therefore, mRNA undergoes a process to convert it into a stable DNA form. This process results in the creation of cDNA (complementary DNA). cDNA is synthesized from RNA using a specific enzyme found in certain viruses, and this enzyme is called reverse transcriptase. This synthesis process is provided by manufacturers, so you can simply follow the manufacturer’s protocol, which is usually very straightforward and easy to perform.

cDNA and qPCR

For example, a product from the Japanese company Takara is commonly used for this process. Takara offers various kits for cDNA synthesis, and their protocols are well-established and easy to follow, making it a popular choice for researchers.

https://www.takarabio.com/products/real-time-pcr/signature-enzymes/reverse-transcriptases/primescript-rt-reagent-kit-%28perfect-real-time%29

The cDNA generated is combined with the primers you designed earlier, and PCR is performed using SYBR Green to measure the amount of PCR product produced. The Ct (Cycle threshold) value is calculated based on the cycle at which the fluorescence signal rises sharply, indicating the presence of the PCR product. To quantify the gene expression, the Ct value of your target gene is compared to the Ct value of a housekeeping gene, such as GAPDH or beta-actin. This allows you to calculate the relative expression of the target gene by comparing the differences in Ct values. Based on this, you can compare the experimental group with the control group to determine any changes in mRNA expression. By examining the differences in the Ct values between the two groups, you can assess whether the target gene’s expression is upregulated or downregulated in response to the experimental conditions.

Even if the mRNA shows a high value, it is recommended to also examine protein-level expression if the gene is truly important. This is because mRNA is easily degraded and can be regulated by various factors, which means its expression might not always correlate with protein expression. By comparing both mRNA and protein expression, you can strongly support your desired hypothesis and provide more reliable evidence for your conclusions.

What is miRNA and siRNA? Limitation and future of miRNA (4)

Watson, Crick and Franklin

Watson and Crick are pivotal scientists in understanding biological pathways. While they are globally renowned, Franklin, who has recently gained recognition, also made a significant contribution to this groundbreaking discovery. Franklin studied the internal structure of DNA using X-ray diffraction and gathered essential data. Notably, the famous “Photo 51” she captured was a critical image that provided strong evidence of DNA’s double-helix structure.

This data is the crucial results to understand DNA structure. This is where a breach of research ethics becomes apparent. Maurice Wilkins, Franklin’s colleague, showed her data to James Watson and Francis Crick without her permission. This data became the critical foundation for Watson and Crick’s 1953 publication of the DNA double-helix model. However, Franklin did not receive proper recognition for her research, which was fundamental to the development of this model. This story remains a case where women’s rights were not properly acknowledged, and research ethics were not clearly established. As a result of this incident, it has led researchers today to adhere to stricter standards in conducting their own studies.

Watson and Crick: DNA structure and central dogma

Although Crick and Watson’s reputation remains controversial, their discovery was remarkable and can be considered the beginning of modern life sciences. The two almost perfectly deciphered the structure of DNA and uncovered key facts, including that DNA consists of two paired strands, the distance between the pairs is 10 Å, and guanine pairs with cytosine while adenine pairs with thymine.

https://www.nature.com/articles/171737a0

In fact, the controversy surrounding their reputation might be somewhat exaggerated, as the two explicitly acknowledged in their paper that Franklin’s experimental results were helpful to their work. They wrote: “We have also been stimulated by a knowledge of the general nature of the unpublished experimental results and ideas of Dr. M. H. F. Wilkins, Dr. R. E. Franklin and their co-workers at King’s College, London.“

(X-ray photographs that merely provided inspiration and were not directly cited in the paper are typically not sufficient to warrant authorship. Acknowledgments, as seen in this case, are the standard practice. Franklin did not receive the Nobel Prize because she passed away before the award was given. This is due to the Nobel Prize being awarded only to living individuals. As a humorous remark, it is often said that the hardest part of winning a Nobel Prize is simply living long enough.)

The achievements of Crick continue to stand as nearly perfect representations of DNA structure, which they accurately detailed in their 1953 Nature paper. Additionally, they discovered the concept of the central dogma, which describes the flow of genetic information from DNA to RNA to protein. “To briefly summarize this paper: DNA replicates through self-division. DNA produces RNA, and RNA produces protein. An exception occurs when RNA is reverse-transcribed into DNA, which is typically observed in RNA viruses.”

https://www.nature.com/articles/227561a0

Since the two papers are not long, I highly recommend reading them.

Does all RNA code for proteins?

Here, RNA is actually more complex. There are numerous different forms of RNA, and not all RNA codes for proteins. This includes tRNA and rRNA, with only mRNA containing the information for proteins. One of the most important types of RNA currently is miRNA, which recently earned a Nobel Prize for its discoverer.

There are numerous other types of RNA, such as miRNA and siRNA. These RNAs are produced by DNA and are made from introns, which do not contain the genetic information from the DNA. This RNA is used in a wide range of biotechnologies, starting from the well-known COVID vaccines to aptamers, which can bind to specific proteins. It is also utilized to create molecules similar to antibodies, but made from DNA or RNA instead of proteins.

I believe this is one of the fields we will see more of in the future.

mRNA and qPCR: Understanding Gene Expression (3)

Discovery of cells

The journey and history of biology begins with the discovery of the cell, a fundamental unit of life. The first cells are reported by Robert Hooke in the 17th century. He invent a microscope and he was curious how the the cork looks like. He interestingly observed the cork consisting of many grids of small cells.  The discovery is reported by the cell wall of plant cells and the plant cell shape is like grids.  His findings marked the dawn of cellular biology, understanding that all living organisms are composed of cells.

Next several years, Antonie van Leeuwenhoek, he used advanced microscope and observed living cells for the first time. He described microorganisms like bacteria and protists, calling them “animalcules. After him,  from 1838 to 1839, Matthias Schleiden (a botanist) and Theodor Schwann (a zoologist) proposed the first two tenets of cell theory: All living organisms are composed of one or more cells. The cell is the basic unit of structure and function in living organisms, as we know. 

The science goes very fast after it. Around 1855, Rudolf Virchow added the third tenet:
all cells arise from pre-existing cells (“Omnis cellula e cellula”). Around 1860s, improved microscopes allowed scientists to study cell organelles, such as the nucleus. Late 1800s, the biologists identified key components like the plasma membrane, cytoplasm, and early versions of what we now call the mitochondria and chloroplasts. The discovery of chromosomes and genes highlighted the role of cells in heredity. Scientists understood cellular respiration and photosynthesis, clarifying how cells generate and store energy.

The discovery of heredity in cells

The cell heredity become famous by Gregor Mendel. He explained the fundamental principles of inheritance while presenting the results of his pea plant crossbreeding experiments.  He discovered the dominant/recessive Gene. 

• Dominant Gene: A dominant gene is one that expresses its trait even if only one copy is present. For example, if a person inherits one dominant gene for brown eyes (B) and one recessive gene for blue eyes (b), the dominant brown eye trait will appear. Dominant traits mask the effect of recessive traits.
• Recessive Gene: A recessive gene only expresses its trait if both copies are present (one from each parent). For instance, to have blue eyes, a person must inherit two recessive genes (bb). If a dominant gene is present, the recessive trait will remain hidden.

Mendel’s laws have become such a renowned theory that they are still used in numerous textbooks today. 

Despite Mendel’s great discoveries in genetics, he did not fully understand why and how inheritance occurs. He also did not fully understand the existence of mutations. The scientists continued to explore and strive to understand the role of cells. Then, a groundbreaking discovery was made: the discovery of DNA by Watson and Crick.

The following information about Watson and Crick will continue in the next story.

Watson, Crick, and Franklin’s DNA Story: Central dogma and RNA (2)