Editor's NoteWith the support of the Shanghai Science and Technology Commission (Project No: 22DZ2304300), The Paper has collaborated with World Science to produce popular science reports on the achievements that have received national and Shanghai science and technology awards.
This report focuses on the first-place project of the 2022 Shanghai Natural Science Award, "Research on the New Functional Mechanisms of RNA Regulation in Spermatogenesis and Male Infertility," led by researcher Liu Mofang from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.
Researcher Liu Mofang from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
In 2006, Liu Mofang concluded her research career at Johns Hopkins University School of Medicine in the United States and returned to China to serve as deputy head of Wang Enduo's research group at the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Liu had been working with RNA for many years and was eager to carve out a new path in the field of RNA.
That year, a significant breakthrough occurred in the RNA field.
Several research teams, including that of Chinese scientist Lin Haifan, discovered a class of novel small RNA molecules in reproductive cells of various model organisms, such as mice and fruit flies. Because these new small RNA molecules function by binding to Piwi proteins in fruit fly reproductive cells, they were named Piwi-interacting RNA (piRNA).
In living organisms, piRNA can be considered a rather "low-profile" class of molecules: first, piRNA is predominantly found in reproductive cells, with virtually no expression in most somatic cells; second, even in reproductive cells, piRNA does not synthesize proteins but exists solely as RNA molecules.
Yet, despite their low profile, piRNAs exhibit remarkable diversity; for instance, more than a million different sequences of piRNA have been identified in mouse reproductive cells.
This raises the question: why does such a wide variety of piRNAs express in reproductive cells? What biological functions do piRNAs actually serve?
Initial studies discovered that many piRNA sequences matched a type of DNA element known as transposons, thus inhibiting the expression of these transposon sequences.
Transposons are a class of molecules capable of "jumping" between different sequences in the genome, and their jumping carries a risk of gene mutations. Therefore, in the long term, they drive evolution. For individual organisms, however, overly active transposons are not beneficial, as the resultant gene mutations can increase the risk of diseases such as cancer. Hence, it was previously found that cells have evolved specific molecular mechanisms to keep transposon activity below a certain threshold.
In the case of reproductive cells, such as sperm cells, which are tasked with accurately passing on genetic information to the next generation, researchers speculate that the "management" of transposon activity here would be even stricter than in other cell types.
The emergence of piRNA perfectly corroborates this hypothesis: piRNA acts like a specialized "security gate" added for reproductive cells, ensuring that transposon activity is suppressed at a lower level.
However, Liu Mofang noticed that not all piRNA molecules could match transposon sequences.
Do these "exceptional" piRNA molecules that do not match transposon sequences still possess unknown biological functions?
Moreover, scientists have found that knocking out the genes responsible for piRNA production or function in model organisms such as mice and fruit flies leads to infertility in those species.
What would happen if piRNA production or function were compromised in humans?
This prompted Liu Mofang to focus her team's research direction on these questions.
Targets Beyond Transposons
In 2010, a French research team discovered that piRNA played a role in degrading the messenger RNA that encodes the developmental factor Nanos during embryonic development in fruit flies. Disruption of the piRNA-binding protein Aub resulted in delayed clearance of Nanos messenger RNA, leading to developmental defects in the embryo's head.
This finding indicated for the first time that, in addition to transposon sequences, messenger RNA that encodes proteins could also be a target of piRNA regulation.
The case discovered in fruit flies further boosted Liu Mofang's confidence in systematically searching for new piRNA targets and functions in mammals.
One interesting peculiarity of piRNA during sperm formation provided a breakthrough for Liu Mofang: During the final transformation of sperm cells into their tadpole-shaped mature form, two peaks of piRNA expression were observed, with piRNAs that did not match transposon sequences primarily expressed during the second peak.
Consequently, Liu Mofang's team first isolated sperm cells from mouse testis tissues at the second peak of piRNA expression and purified the complexes containing piRNA. Deep sequencing revealed that while over two-thirds of the paired RNA molecules matched transposon sequences and were classic piRNA, around 20% were actually messenger RNA capable of encoding proteins.
Next, they selected ten pairs of complementary piRNA and messenger RNA to verify their physical interactions and demonstrated through functional studies that the existence of specific piRNA promotes the degradation of the corresponding messenger RNA.
What is the significance of this discovery?
It turns out that piRNA promotes the degradation of messenger RNA primarily during the transition stage when sperm cells change from a round to an elongated rod shape. For mature sperm to ensure sufficient mobility, they need to expel materials that could add weight. It's akin to a Shenzhou spacecraft, which sheds booster rockets with excess weight during launch, ultimately leaving just a "big iron ball" carrying the astronauts into space. In the process of sperm formation, cells transform from a round shape to elongated and finally to a tadpole shape, necessitating the clearance of most messenger RNA from the cells.
Liu Mofang's team's work indicates that piRNA plays a crucial role in helping sperm cells lighten their load.
In other words, during specific stages of sperm formation, piRNA also takes on the honorable mission of being a "cleaner."
Hardworking piRNA: Beyond Cleaning, Also Responsible for Some Production Work
Liu Mofang's team has selected over 100 pairs of piRNA and messenger RNA for validation, and most of these piRNA promote the degradation of messenger RNA, resulting in decreased levels of the corresponding encoded proteins.
However, there were five exceptional pairs: Researchers were surprised to find that the expression of these piRNA did not affect the levels of the corresponding messenger RNA. For the encoded proteins, piRNA expression not only did not lower protein levels but actually increased them!
This bizarre phenomenon raises the question: Is it a result of experimental error, or does it hint at an unknown new biological principle?
The Liu Mofang team did not dismiss these five pairs of unexpected "exceptions." Through in-depth investigation, they ultimately provided answers to another significant question in the field of sperm development.
The phenomenon of "transcription-translation uncoupling" has long puzzled researchers in the field of spermatogenesis: during development, sperm cells must compress their nuclei, which carry genomic information, to "shed weight," but this process halts the transcription of messenger RNA. To ensure that sufficient proteins are produced in the later stages of sperm development, sperm cells pre-synthesize the required messenger RNAs and store them in a complex known as "messenger ribonucleoprotein" (mRNP). The mRNP acts like a temporary "warehouse" for the cell, with the messenger RNA stored in a "dormant" state, and the activity of translating proteins is very low.
When sperm development progresses to the stage where the shape transforms from round to elongated, the messenger RNA stored in mRNP is released in an orderly manner, rapidly translated to produce the proteins required for the later stages of sperm development.
This mechanism of transcriptionally synthesizing messenger RNA first, then not immediately translating proteins but saving them in mRNP complexes until the time is right to reactivate translation is referred to as "transcription-translation uncoupling."
Liu Mofang's team noted that among the five piRNAs that could unexpectedly upregulate protein levels, two proteins encoded were known to be highly important for late sperm development.
Consequently, they speculated whether piRNA could be the pivotal molecule that encourages the release of mRNP to activate messenger RNA translation.
Indeed, specific piRNA can recruit a series of translation regulators by binding to a special sequence at the end of the messenger RNA, activating the translation of that messenger RNA.
Furthermore, they found that this new mechanism is not limited to the five previously identified piRNA; proteomic experiments revealed that this piRNA-dependent translation activation mechanism can be extended to hundreds of different proteins.
This finding further solidifies piRNA's status as a "model worker" in reproductive cells: they first repress transposon activity throughout the process of sperm development to ensure genomic stability; secondly, they reactivate the translation activity of messenger RNA stored in mRNP; finally, they ensure the clearance of messenger RNA in the late stages of sperm development, allowing the final mature sperm to be light and agile, which enhances its motility.
In summary, Liu Mofang's team's series of studies uncovered that piRNA has a dual regulatory role concerning messenger RNA in sperm cells.
Translational Activation Ally: The Phase-Separating Protein FXR1
With the clarification of piRNA's role in the phenomenon of "transcription-translation uncoupling," Liu Mofang's team became increasingly interested in the dynamic changes of mRNP complexes during different stages of sperm development.
But how do sperm cells know when to reactivate protein translation?
To tackle this question, Liu Mofang's team purified ribosome complexes from the testes of juvenile and adult mice in a state of high activity and identified twelve translation regulatory factors enriched in adult mouse testes using proteomics methods. Among them, a protein named FXR1 garnered particular attention from the researchers.
As sperm cells undergo morphological changes, the expression levels of FXR1 gradually increase. Moreover, FXR1 can bind to thousands of different sequences of messenger RNA and promote the translation of the corresponding proteins. Interestingly, a homolog of FXR1 possesses a unique "phase-separation" property—at higher concentrations, it can form special structures that aggregate within cells.
So, does FXR1 also have a similar phase-separation property?
Experiments in vitro demonstrated that, with increasing concentration, FXR1 indeed forms structures characteristic of phase separation. Additionally, knocking out FXR1 in reproductive cells resulted in infertility symptoms in male mice.
Thus, Liu Mofang's team proposed a bold hypothesis: during sperm development, the increase in FXR1 protein levels might cluster dispersed mRNP complexes through phase separation, thereby lifting the repression of translation activity by the mRNP complexes and prompting sperm development to the next stage.
To test this hypothesis, they designed an impressive experiment: through mutagenesis screening, the researchers identified a variant of FXR1. This variant differed from the original FXR1 by only one critical point mutation in the amino acid sequence, retaining the ability to bind to RNA molecules but losing its phase-separation capability. When the FXR1 protein in mouse spermatogenic cells was replaced with the mutant version, the researchers excitingly found that the translation activation ability of the mRNP complexes significantly decreased.
Clearly, FXR1 is likely regulating the activation of translation in sperm cells through the mechanism of phase separation.
This discovery innovatively explains the crucial role of phase separation in the process of sperm development. On May 25, 2021, Liu Mofang's team submitted their manuscript to Science.
However, due to the significance of this finding, reviewers raised many careful questions regarding the details of the arguments. Interestingly, one reviewer noted in their comments: “Even if the phase-separation model proves to be incorrect in the future, the findings of this work are novel and important. The researchers have done a lot of work, and if my comments delayed the publication of this work, I apologize…”
After ten months of meticulous revisions, Liu Mofang's team resubmitted the paper, which included more supplementary experiments, to Science for review on March 23, 2022, and it was eventually published on August 12, 2022.
Building on Success
From 2006 to the present, Liu Mofang's team has steadily advanced in the field of piRNA, continuously deepening our understanding of the regulatory mechanisms of sperm cell development.
At the same time, they are also focused on how basic research can assist clinical practice. One significant contribution published in Cell