Author: Margarita Angelova

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Take a look in the past to foresee the future: the evolution of genome engineering

Transposons, also known as mobile genetic elements, are segments of DNA capable of moving within the genome. This mobility can potentially induce mutations, as transposon “jumping” within a gene may interrupt the gene and trigger its loss. Transposons utilize their own enzymes, transposases, to move in and out of the genome.

Due to the potential harm caused by transposons, cells continually evolve strategies to control them. In response, transposons adapt, arming themselves with new strategies to evade cellular control. Mobilized transposons can hijack adjacent DNA information and transfer it to a new location, sometimes involving a gene that provides a survival advantage, thus increasing the transposon’s chances of propagation. Simultaneously, cells “steal” genes from transposons, repurposing them to function in processes advantageous to the cell. This ongoing arms race between transposons and host defense systems has facilitated a widespread exchange of genes between different life forms and is a major driver of genome evolution across all domains of life. Transposons play a crucial role in this process.

In bacteria, the CRISPR-Cas9 system is fundamental for protection against viral infections. Using a small RNA molecule called guide RNA, the CRISPR-Cas9 system acts as a nuclease, akin to DNA cutting scissors, destroying the viral genome. This system can be modified to edit genes in organisms beyond bacteria. The guide RNA is altered to direct the Cas DNA scissor to a gene of interest instead of a viral genome. This programmable gene-editing system has diverse applications, from basic biology research to disease treatments, and it earned the 2020 Nobel Prize in Chemistry for Emmanuelle Charpentier and Jennifer Doudna. Notably, scientists have recently traced the evolutionary origins of CRISPR-Cas9 to transposons.

In a recent study published in Nature, Columbia postdoc Chance Meers and his colleagues provided further insight into the evolution of the CRISPR-Cas9 nuclease system by examining how transposons use cellular nucleases to proliferate within genomes. They focused on a specific transposon system called insertion sequences (IS), which are bacterial transposons encoding only the genes necessary for transposon excision and movement to a new genomic location. This includes a transposase. However, many IS transposons contain an accessory gene that carries the information for a nuclease which functions similarly to the CRISPR-Cas system as an RNA-guided DNA cleaving enzyme.

The IS element does not need a nuclease for its movement, the transposase is sufficient to mobilize the transposon to a new location. So why are so many of the IS elements carrying those RNA-guided DNA scissors? While the mechanism by which IS elements are mobilized by their transposase is similar to a cut-and-paste process, leaving no copy at the original DNA location, Meers and colleagues discovered that the rate of excision is more efficient than the rate of integration. Without an additional system for transposon proliferation, the transposon would eventually be lost since not every excised copy manages to be reintegrated. The study of the authors revealed that the accessory CRISPR-like functioning nuclease guides a copy of the IS element back to its original location, generating two copies of the element—one at the original site and one at the newly inserted site. This changes the mechanism from cut and paste to cut and copy when the transposase function is complemented by the accessory nuclease, increasing the transposon copy number present in the genome, and thus serving for the transposon selfish proliferation (Figure 1).

Figure 1. RNA-guided nucleases assist transposon survival via guiding specific breaks at donor sites and transposon restoring repair. During DNA replication the two strands of the DNA duplex are separated and each strand serves as a template for the synthesis of a new DNA molecule. Single stranded DNA facilitates the transposition mediated by the transposase of IS elements. The excision of the transposon leads to its loss at one of two DNA strands, while DNA replication restores the transposon on the other DNA strand. Simultaneously, transposon excision restores a target site at the transposon donor location which is specifically recognized by a guide RNA (blue segment). This guide RNA directs the accessory nuclease on the excision location and DNA cleavage occurs. The resulting double strand break (DSB) is lethal for the bacteria and its only chance of survival is to copy the information from the homologous newly synthesized intact DNA molecule that contains the transposon. This leads to reconstitution of the transposon at the donor site on the two newly synthesized DNA duplexes. Transposition at the new target sites will produce new guides, specific for the new insertion site location (orange segment), which will facilitate the future transposon spread and maintenance by identical mechanism. 

By developing powerful assays to track the movement of transposons within bacterial genomes, or other small DNA molecules called plasmids, as well as from one bacterium to another, Meers and his colleagues uncovered how those RNA-guided DNA cutting nucleases work. The authors’ discovery enhances our understanding of how proteins collaborate with RNA guides to target and edit genomes. In addition, the study unveils the original role of CRISPR’s nucleases from an evolutionary perspective before they were repurposed by the bacterial host genome to fight viruses, which was to serve the selfish propagation of transposons. Moreover, given the abundance of transposons in genomes, it is highly likely that other systems different from CRISPR-Cas9 and derived from transposon genes exist, waiting to be discovered and potentially harnessed, expanding the biotechnological tools available for programmable and specific genome engineering. Thousands of ancient transposons in bacterial genomes carry RNA-guided DNA nucleases that can potentially be programmed to cut DNA similarly to the CRISPR-Cas9 system.

Reviewed by: Trang Nguyen, Giulia Mezzadri

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Copy & Paste – an essential mechanism to repair damaged DNA

Maintaining genome integrity is crucial for cell survival and genome instability is one of the hallmarks of cancer. The genomes of many living organisms including humans are composed of different numbers of DNA molecules that are folded into structures called chromosomes. Each chromosome represents one double stranded DNA molecule. Most of our cells harbor two sets of each chromosome, which are denoted as homologous chromosomes (Figure 1, left panel). The homologous chromosome pairs can contain identical or different versions (alleles) of the genes that they carry since they are inherited from each parent. In this sense homologous chromosomes are genetically non-identical.

Most of the cells in our body are constantly renewed and replaced in a process that involves cellular division. During cell division the genome needs to be duplicated so that each of the newly formed daughter cells receives a genome copy. The process of DNA duplication is called DNA replication. During replication, each of the two strands of the DNA double helix serves as a template for the synthesis of a new complementary DNA strand. These chromosome copies that are produced during cell division are named sister chromatids (Figure 1, right panel), and they are found transiently in the cells just before the division. Unlike the homologous chromosomes, the alleles of the genes in the sister chromatids are genetically identical since they are produced by DNA replication.

Figure 1: Schematic representation of the homologous chromosomes and the sister chromatids produced during cellular division. Created with BioRender.com

During DNA replications, as well as under the influence of diverse environmental and endogenous agents, lesions constantly occur on DNA. These DNA “injuries” can trigger mutations, compromise the genome integrity, or even cause cell death. DNA double-strand breaks (DSBs) are one of the most deleterious types of DNA lesions that can lead to gross chromosome rearrangements. Similar rearrangements are very common in cancer.  One of the major mechanisms for DNA DSBs repair is homologous recombination (HR), a process explained below. Mutations in different genes from the HR pathway have been associated with diseases like cancer of Fanconi anemia, as well as to hypersensitivity to DNA-damaging agents which increase mutation accumulation.

One of the versions of HR involves the use of the intact sister chromatid as donor of information. Since the sister chromatid has an identical sequence to the damaged DNA molecule, this repair system faithfully restores the genetic information and is considered as being error free. Homologous recombination can also occur between the homologous chromosome pairs in the case of non-dividing cells. In HR, the DNA DSB ends are processed and a long single-stranded DNA (ssDNA) overhang is left to serve as platform for the assembly of the protein machinery mediating the repair (Figure 1, a). This ssDNA is coated by the protein RPA to prevent degradation and folding. Subsequently RPA is replaced by RAD51, into a structure termed the presynaptic nucleoprotein filament. This filament is capable of searching for a homologous undamaged DNA molecule that will serve as a template for repair. This exchange between RPA and RAD51 on ssDNA in the presynaptic filament assembly is facilitated by mediators of HR. One of those mediators is a complex of four proteins that are paralogues of RAD51: the BCDX2 complex. However, the exact mechanism of action of the BCDX2 complex in HR remained elusive.

Figure 2. Double-strand breaks (DSBs) can be repaired by several homologous recombination (HR)-mediated pathways. Represented is a simplified version of the “synthesis-dependent strand annealing HR pathway”. In all pathways, the repair is initiated by resection of a DSB to provide 3′ single-stranded DNA (ssDNA) overhangs (a and b). The ssRNA is rapidly coated with RPA to prevent damage and folding (c). RPA is exchanged with RAD51 with the help of HR mediator complexes, which are composed from different RAD51 paralogues, one of which is four protein complex BCDX2 (d). RAD51 coated DNA can search for the non-damaged homologous chromosome / sister chromatid pair and invade that intact DNA duplex (e). After strand invasion and “copying” of the information from the intact DNA on one of the damaged strands from the blue DNA duplex, the reaction can proceed to hybridization to the ssDNA on the other break end of the blue DNA duplex, followed by DNA synthesis (f) and restoring  of blue DNA molecule (g). Created with BioRender.com

Recent studies from Columbia postdoc Aviv Meir and colleagues revealed the structure of the human BCDX2 complex. Cryogenic electron microscopy, a technique that allows the high-resolution structure determination of biomolecules in solution, was used to resolve both the  free and single-strand DNA-bound states of BCDX2. This provided the first structural information of one of the RAD51 paralogues complexes. This structural information provides insight into how the complex assembles and disassembles, which in turn is linked to the regulation of its function. The scientists also discovered by single molecule analysis that the association of BCDX2 with RPA–ssDNA enhances the rate of assembly of the RAD51–ssDNA filament. In humans, BCDX2 binds the RPA–ssDNA prior to the arrival of RAD51 and then promotes the RAD51 filament assembly. This novel mode of action for the proteins of the BCDX2 complex is different from what was previously observed in other organisms, where BCDX2 only transiently associates with the ssDNA. The work by Dr. Meir and colleagues, recently published in the prestigious journal Nature, not only elucidates how BCDX2 mediates RPA–RAD51 exchange on ssDNA but also provides a foundation for deciphering how alterations in BCDX2 subunits that were found in patients with cancer can impact genome repair and can lead to the pathogenesis. This valuable information opens the doors for future targeting of those “defectuous” BCDX2 parts for therapeutic developments.

Reviewed by: Maaike Schilperoort, Apurva Limaye, Giulia Mezzadri , Trang Nguyen

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​How a virus invading a cell limits another virus access to the same cell

Alphaviruses can infect both vertebrate and invertebrate animals.  Their transmission between species and individuals occurs mainly via mosquitoes. These viruses are small, spherical, and have a genome composed of a single strand ribonucleic acid (RNA) in the “positive-sense”. The alphaviral life cycles and their RNA genome amplification (replication) have been studied since their discovery in 1953. However, the very initial events of viral genome replication have remained unknown.

Positive-strand RNA viruses genome can be directly translated into viral proteins with the participation of factors and structures provided by the invaded host cell. However, in order to amplify the viral genome and to produce new viral particles during the virus propagation, the positive RNA strand has to be converted to its complementary negative strand by an enzyme that is encoded in the viral genome. This enzyme uses RNA as a template to synthesize RNA, a so-called RNA-dependent RNA polymerase (RdRp). RdRp are used during replication of the genome to synthesize a negative-sense antigenome that is then used as the template to create a new positive-sense viral genome, necessary for the future viral progeny and viral propagation (Figure 1).

Figure 1. Overview of the alphavirus life cycle. Alphaviruses enter the cell by recognizing a cell receptor, followed by release into the host cell of the viral plus-strand genome (1). The genome serves as a template carrying the information for production of a fused version of viral proteins (viral polyprotein, 2). This polyprotein is cleaved to different combinations (not shown) constituting an RNA-dependent RNA polymerase, and two forms of protein complexes required for viral replication (3 and 4). The consecutive cleavage of the polyprotein has been shown to influence transitions in production between the full-length minus-strand RNA, the genomic plus strand, as well as of another form of viral RNA (not shown) required for subsequent viral particles (nucleocapsid) assembly and release (5 and 6). Figure adapted from the original paper.

A phenomenon known as superinfection exclusion has been previously observed, where infection by one virus can block the infection of a subsequent homologous virus. This form of viral competition protects the virus to complete its reproduction without the need to share the cell’s resources with homologous viruses or with its own progeny. One of the mechanisms of superinfection inclusion can be by reducing the host cell receptors that the virus uses to recognise and enter into the cell. However, such changes in the cells are thought to take place several hours upon infection and for some viruses the phenomenon of superinfection exclusion has been observed as soon as just 15 minutes of the first infection (Figure 1). This rapid competitive behavior was observed over 40 years ago. This mechanism providing such rapid protection over a secondary infection is very beneficial, especially considering the ability of the virus to enter cells within minutes. However, its causes, as well as the very earliest stages of alphaviral replication and whether the two processes are linked has remained unclear.

Previous studies of the alphavirus’s life cycle have mainly used populations of infected cells. The use of recently developed single cell-based methods allows to overcome several limitations of population-level studies. For example, the classic population-based studies have shown the average growth of the virus over time across millions of cells and have revealed that the first release of viral progeny can be detected as early as 3–4 hours post infection (Figure 1). However, there is an inherent cell-to-cell variability in the infection spreading in a group of cells. Use of single-cell analyses in biology has shown how the variability of individual cells can be masked by the overall population’s behavior and how variability between individual cells contribute to viral growth and spreading kinetics. An important challenge on how the dynamics of early replication could affect the competitive interactions is the lack of sensitivity on low-abundance targets during early infection. In order to capture the dynamics of the earliest stages of replication, it is necessary to utilize an approach with sufficient sensitivity to simultaneously measure individual molecules of multiple viral RNA species at low abundance.

The recent work published from Columbia postdoc Zakary Singer and his colleagues presents a new quantitative detailed characterization of the initial replication activity of members of the alphavirus genus, Sindbis virus. The study consists in analyzing the viral genome biology at the level of individually affected cells and not in a group of cellular population. The authors used quantitative live single cell imaging technique to follow and measure the viral replication in real time upon infection as well as to elucidate how these contribute to the rapid exclusion of a superinfecting alphavirus. Singer and colleagues observed that the rapid onset of viral RNA synthesis as a passive superinfection exclusion mechanism could contribute to this advantage. Furthermore, a mathematical model of exponential viral growth in a resource-limited environment appeared consistent with the measurements of viral replication. The authors also investigated whether there is a bidirectional inhibition between two viruses in the same cell, by experimental measurements and a mathematical modeling of competitive growth using parameters estimated from single-virus infection experiments. The results from both methods suggested that the superinfecting virus is equally able to reduce replication levels of the first virus and that the cell appears to have fixed carrying capacity that sets up the combined replication level of the two viruses. Due to the speed of Sindbis replication would strongly disadvantage the second virus and reduce the second virus’s replication, showing the importance of intrinsic growth kinetics in alphaviral superinfection exclusion.

The work by Singer and colleagues also allowed to shed light on classic questions remaining in alpha virology and suggested a revised model of early replication wherein both plus- and minus strands are made at a similar rate during early infection in contrast to previous claims that initially the positive strand RNA production is predominant. Additionally, the paper provides one of the earliest detections of alphaviral replication, as well as a new framework for understanding early replication and the resulting exclusionary phenomenon. Finally, the work hints on how in the future the complex interplay with innate immunity and stochasticity will be broadly relevant to the study of many infectious diseases, and how quantitative models might lead to improved antivirals. Check out more from the original publication.

Edited by: Sam Rossano and Trang Nguyen

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A continuous war between DNA elements shapes genome evolution

While genomes include the totality of genes that determine an organism’s biological identity, genes can only be a tiny fraction of the genome. In humans, and in many other species, DNA contains multiple diverse regions. One example are transposons, or mobile genetic elements, which are parts of DNA that can move in new places in the genome (Figure 1). The “genomic walks” of transposons are potentially harmful, since when they “jump” within a gene, they trigger mutation and loss of that gene. Cells are constantly evolving new strategies to keep transposons under control. In response, transposons adapt and arm themselves with new strategies to escape cellular control.

Sometimes, a mobilized transposon hijacks additional DNA information that it transfers to the new location with itself. When this additional DNA happens to be a gene providing survival advantage, not only the recipient increases its gene pool and survival capacities but the transposon also increases its chances to propagate. This process of transmission of information between different species is termed horizontal gene transfer and represents a major driver of genome evolution across all domains of life. Transposons have been key players in this process.

Figure 1. Schematic representation of a DNA transposon and its movement between two DNA molecules. Image created with BioRender.com.

Transposons can be considered as reminiscent of ancient viral infections that managed to integrate in a host cell genome but later lost the capacity to completely exit the cell. Bacteria have evolved a fascinating mechanism to remember viruses that they have already encountered. This acquired immunity involves a family of DNA sequences named CRISPR and their associated protein partners Cas. The CRISPR sequences are fragments of DNA derived from different viruses that have infected the bacteria and were integrated in the bacterial genome, creating a footprint of previous infections (Figure 2, upper part). When cells are infected, they use the CRISPR catalog and compare it to the invading DNA, helping it to recognize recurrent viruses and destroy them more rapidly. Cas proteins participate in the degradation of the viral DNA. (Figure 2, lower part).

Advances in research showed that the CRISPR-Cas complexes can be modified to edit genes in different organisms. To do this, part of the complex is changed to lead the Cas protein to a gene of interest, instead towards a viral genome. This system of gene editing has already found numerous important applications ranging from basic biology research to disease treatments and development of new technologies. The discovery of CRISPR-Cas was acknowledged with the 2020 Nobel Prize in Chemistry to Emmanuelle Charpentier and Jennifer Doudna.

Figure 2. Schematic representation of the CRISPR-Cas9 adaptive immune system of bacteria. Briefly, upon infection the viral DNA is fragmented and part of it is integrated in a special region of the bacterial genome, the CRISPR locus. CRISPR sequences get copied into shorter RNA molecules that carry parts of sequences identical to the sequence of a certain virus. Once copied, these short RNAs form complexes with Cas proteins and serve as “guides” for them towards potential complementary viral DNAs. The viral DNA is destroyed by the Cas protein if it can hybridize with the short RNA from the CRISPR locus. Image created with BioRender.com. 

Interestingly, researchers have found an intriguing interconnection between transposons and the  CRISPR–Cas defense system. In a striking example a bacterial transposon has hijacked some of the genes of a CRISPR-Cas system and uses those genes for its own propagation in genomes. These transposons are called CRISPR-associated transposons. Such widespread exchange of genes is caused by the never-ending arms race between the transposons and their hosts’ defense systems. In the recent publication of Hoffman and colleagues, five Columbia postdocs Minjoo Kim, Leslie Y. Beh, Jing Wang, Diego R. Gelsinger, and Jerrin Thomas George collaborated and brought significant insight in the functioning of one CRISPR-associated transposon.

In their publication, the authors monitored in detail the formation of  this RNA-guided transposon and its associated complexes, which enabled them to resolve distinct protein recruitment events that take place before the integration of the transposon. They also found that even if initially hundreds of non-desired genomic sites are targeted for integration at the end only few of those sites recruit the whole transposon machinery that is required for integration at this genomic location. This discovery offered insights into how the potential target sites in the host genome are identified, screened and approved for integration, allowing the transposon system to be specific.

To advance the understanding of interactions responsible for the assembly of the transposon associated proteins, the authors determined the structure of one of the interacting proteins, named TnsC and found that it is forming rings of seven molecules of TnsC that can pass DNA through the central pore of the ring. This helps to correctly position DNA for the following integration (Figure 3). The resolved molecular structure also allowed to gain clarity on how TnsC mediates the communication between the proteins in this transposon complex that are responsible on the one hand for the targeting and on the other hand for the integration at a specific genomic location. Their results pointed to TnsC as the proofreading checkpoint that ensures the specific selection of genomic sites for transposition.

Figure 3. Upper part: Model of the 7 molecules of TnsC forming a ring through which DNA is passed. Lower part: Representative experimental result from Hoffmann et al. showing different configurations and views of the DNA-TnsC complex. Figure adapted from the original publication. 

In summary, the paper not only deciphers the molecular specificity of consecutive factor binding to genomic target sites in this interesting process of RNA-guided transposition, but the resolved detailed structure also provides valuable information for the development of future biotechnologies in the field of programmable and specific integration of DNA in desired genomic locations. Such technology differs from the above-described original CRISPR-Cas system currently used for genome editing, because it has the potential to be less mutagenic, as well as because it provides the opportunity to insert much longer pieces of DNA in a desired location. RNA-guided transposases hold tremendous potential for future biotechnological and human therapeutic applications and will without a doubt accelerate novel discoveries. Find out more in the original publication.

Reviewed by: Trang Nguyen & Maaike Schilperoort

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What can we do to enter a new era in antimalarial research? A promising story from genetics to genomics.

Plasmodium falciparum is a unicellular organism known as one of the deadliest parasites in humans. This parasite is transmitted through bites of female Anopheles mosquitoes and causes the most dangerous form of malaria, falciparum malaria. Each year, over 200 million cases of malaria result in hundreds of millions of deaths. Moreover, P. falciparum has also been involved in the development of blood cancer. Therefore, study of malaria-causing Plasmodium species and the development of anti-malarial treatment constitute a high-impact domain of biological research.

Antimalarial drugs have been the pillar of malaria control and prophylaxis. Treatments combine rapid compounds to reduce parasite biomass with longer-lasting drugs that eliminate surviving parasites. These strategies have led to significant reductions in malaria-associated deaths. However, Plasmodium is constantly developing resistance to existing treatments. The situation is further complicated by the spread of mosquitoes resistant to insecticides. Additionally, asymptomatic chronic infections serve as parasite reservoirs and the single candidate vaccine has limited efficacy. Thus, the fight against malaria requires sustained efforts. A detailed understanding of P. falciparum biology is still crucial to identify and develop novel and efficient therapeutic targets.

Recent progress in genomics and molecular genetics empowered novel approaches to study the parasite gene functions. Application of genome-based analyses, genome editing, and genetic systems that allow for temporal regulation of gene and protein expression have proven to be crucial in identifying P. falciparum genes involved in antimalarial resistance. In their recent review, Columbia postdoc John Okombo and colleagues summarize the contributions and limitations  of some of these approaches in advancing our understanding of Plasmodium biology and in characterizing regions of its genome associated with antimalarial drug responses.

P. falciparum requires two hosts for its development and transmission: humans and Anopheles mosquito species. The parasite life cycle involves numerous developmental stages. In humans take place stages of Plasmodium’s development that are part of its “so-called” asexual development. On the other hand, mosquitos harbour other stages of the parasite development, associated with its sexual reproduction (Figure 1). Humans are infected by a stage called “sporozoites” upon the bite of an infected mosquito. Sporozoites enter the bloodstream and migrate through to the liver where they invade the liver cells (hepatocytes), multiply and form “hepatic schizonts”. Then, the schizonts rupture and release in the circulation the stage of “merozoites” which invade red blood cells (RBCs).  The clinical symptoms of malaria such as fever, anemia, and neurological disorder are produced during the blood stage. In RBCs are formed “trophozoites”, that have two alternative paths of development. They can either form “blood-stage schizonts” that produce more RBC-infecting merozoites or can alternatively differentiate to sexual forms, male and female “gametocytes”. Finally, gametocytes get ingested by new mosquitoes during blood meal where they undergo sexual reproduction forming a “zygote”. The zygotes then pass through several additional stages until maturation to a new generation of sporozoites, closing the parasite life cycle (Figure 1).

Figure 1: Life cycle of Plasmodium falciparum. Image created with BioRender.com

This complexity of the Plasmodium life cycle presents opportunities to generate drugs acting on various stages of its development. The review of Okombo and colleagues underlines how new genomic data have enabled the identification of genes contributing to various parasite traits, particularly those of antimalarial drug responses. The authors recap genetic- and genomic-based approaches that have set the stage for current investigations into antimalarial drug resistance and Plasmodium biology and have thus led to expanding and improving the available antimalarial arsenal.

For instance, in “genome-wide association studies” (GWAS), parasites isolated from infected patients are profiled for resistance against antimalarial drugs of interest, and their genomes are studied in order to identify genetic variants associated with resistance. In “genetic crosses and linkage analyses”, gametocytes from genetically distinct parental parasites are fed to mosquitoes in which they undergo sexual reproduction. The resulting progeny are inoculated into humanized human liver-chimeric mice-models that support P. falciparum infections and development. The progeny is later analyzed to identify the DNA changes associated with resistance and drug response variation. In “in vitro evolution and whole-genome analysis” antiplasmodial compounds are used to pressure P. falciparum progeny to undergo evolution to drug-resistant parasites. Their genome is then analyzed to identify the genetic determinants that may underlie the resistance. “Phenotype-driven functional Plasmodium mutant screens” are based on random genome-wide mutation generation and selection of mutants that either are resistant to drugs or have affected development, pathogenicity, or virulence. Such an approach has also led to the discovery of novel important genes. In addition, the review covers a number of cutting-edge methods for genome editing used to study antimalarial resistance and mode of action. Experiments using genetically engineered parasites constitute a critical step in uncovering the functional role of the identified genes. Finally, the reader can also find an overview of Plasmodium “regulatable expression strategies”. These approaches are particularly valuable in the study of non-dispensable (essential) genes. Additional information on other intriguing and powerful techniques are further described in the original paper.

Article reviewed by: Trang Nguyen, Samantha Rossano, Maaike Schilperoort

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Let’s get MDM2 and MDMX out of the shadow of p53

When it comes to cancer, one molecule stands out as being among the most extensively studied: the p53 tumor suppressor protein. p53 can exist in cells in several different forms. When p53 is in its so-called wild-type form, it is capable of activating various responses that contribute to tumor suppression. In their recent review, Columbia postdoc Rafaela Muniz de Quieroz and colleagues summarize the vast scientific literature on two key regulators of p53: MDM2 and MDMX. Both MDM2 and MDMX are known to interact with p53 and disrupt its function. Their absence has been linked not only to increased cancer development, but also to a number of dysfunctions, including embryonic lethality in mice. MDM2 has been shown to negatively regulate p53 by diverse mechanisms spanning from expression of the p53 gene to degradation of the p53 protein or its expulsion from the cellular nucleus, where the protein accomplishes its function. Although very similar to MDM2, MDMX is less well studied. We do know, however, that MDMX is a protein that can work together with the MDM2  in p53 degradation.

While many reviews and studies have pointed to the roles of MDM2, and to a lesser extent of MDMX, in p53 regulation, the current review by Quieroz and her colleagues  puts a larger focus on the myriad of p53-independent activities of MDM2 and MDMX. The authors provide important details about the p53-independent functions of both MDMX alone and as part of a MDM2–MDMX complex. The review discusses some key features in the structure and function of the proteins, including  key parts  that are relevant for their function, for some associated abnormalities, or for the formation of MDM2 and MDMX complexes.

MDM2 and MDMX are regulated on multiple levels within cells. These include regulation on the DNA level, including usage of several alternative promoters (DNA sequences needed to turn a gene on or off). One of the promoters of MDM2 and MDMX is regulated by their target p53, but there are also p53-independent promoters capable of switching on the genes of MDM2 and MDMS regardless of p53. In addition, numerous variations in the DNA sequences, the so-called single nucleotide polymorphisms (SNPs), affect the expression of the two genes and are relevant to different pathologies. Regulation on the RNA level includes co-transcriptional regulation like alternative splicing, as well as post-transcriptional regulation by microRNAs, long non-coding RNAs, circular RNAs, or RNA binding proteins. The review also presents a detailed characterization of the regulation of MDM2 and MDMX at the protein level, by summarizing data on numerous post-translational modifications or interacting partners of the two proteins, with regards to the different p53 contexts of the cited studies. Amongst the presented binding partners are some of the more recently identified interactors of the MDMs, which include proteins involved in the defense against several viruses. Overall, both MDM2 and MDMX stand out as extensively regulated at virtually every known level which according to the authors “attests to their relevance not only as inhibitors of p53 but of myriad other cellular activities and outcomes on their own”.

Since MDM2 and MDMX have majorly been studied in their relation to inhibit wild-type p53, of a particular interest stands a section of the review summarizing numerous processes in which the two proteins have been shown to be involved in cells lacking wild-type p53 (Figure 1).

Figure 1: Nonmalignant disease (left) and cancer-related (right) p53-independent functions of MDM2 and MDMX (adapted from Figure 4 of the review).

As shown in Figure 1, the p53-independent roles of MDM2 and MDMX in cancer and in other pathologies are versatile. That hints to the importance of uncovering molecules that can modulate the deleterious effects associated with dysfunctions of the two MDMs. A summary of numerous molecules that were shown to regulate the two proteins and thus consist of potential therapeutic targets, are also discussed in the review. Again the authors put an emphasis on how such small molecules might be useful in cells that lack wild-type p53. This is important not only because the two proteins have multiple functions other than regulating wild-type p53 which can be studied in such cells, but also because an important percentage of tumors is characterized by absence of wild-type p53.

The last section of the review points out some outstanding questions and directions for future research. If the fascinating questions of the versatile p53-independent roles MDM2 and MDMX have sparked your interest, find out more from the original paper.

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