Near-Infrared Luciferase Complementation Assay with Enhanced Bioluminescence for Studying Protein-Protein Interactions and Drug Evaluation Under Physiological Conditions.

Identification of protein-protein interactions (PPIs) that occur in various cellular processes helps to reveal their potential molecular mechanisms, and there is still an urgent need to develop the assays to explore PPIs in living subjects. Here, we reported a near-infrared split luciferase complementation assay (SLCA) with enhanced bioluminescence produced by cleaving a luciferase, Akaluc, for exploring and visualizing PPIs in living cells and live mice. Compared with the previously developed and widely used red SLCA based on split firefly luciferase (Fluc-SLCA), the signal intensities for PPI recognition in living cells and live mice of the Akaluc-SLCA increased by ∼3.79-fold and ∼18.06-fold in the measured condition, respectively. Additionally, the interactions between the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular RNA processing proteins were identified, and the drug evaluation assays were also performed in living cells using Akaluc-SLCA. This study provides a new tool in the near-infrared region for the identification of PPIs in living cells and in vivo and new information for the understanding and treatment of SARS-CoV-2.

The landscape of chimeric RNAs in non-diseased tissues and cells.

Chimeric RNAs and their encoded proteins have been traditionally viewed as unique features of neoplasia, and have been used as biomarkers and therapeutic targets for multiple cancers. Recent studies have demonstrated that chimeric RNAs also exist in non-cancerous cells and tissues, although large-scale, genome-wide studies of chimeric RNAs in non-diseased tissues have been scarce. Here, we explored the landscape of chimeric RNAs in 9495 non-diseased human tissue samples of 53 different tissues from the GTEx project. Further, we established means for classifying chimeric RNAs, and observed enrichment for particular classifications as more stringent filters are applied. We experimentally validated a subset of chimeric RNAs from each classification and demonstrated functional relevance of two chimeric RNAs in non-cancerous cells. Importantly, our list of chimeric RNAs in non-diseased tissues overlaps with some entries in several cancer fusion databases, raising concerns for some annotations. The data from this study provides a large repository of chimeric RNAs present in non-diseased tissues, which can be used as a control dataset to facilitate the identification of true cancer-specific chimeras.

A cell-based splicing reporter system to identify regulators of cis-splicing between adjacent genes.

Chimeric RNAs generated by cis-splicing between adjacent genes (cis-SAGe) are increasingly recognized as a widespread phenomenon. These chimeric messenger RNAs are present in normal human cells, and are also detected in various cancers. The mechanisms for how this group of chimeras is formed are not yet clear, in part due to the lack of a tractable system for their experimental investigation. Here we developed a fast, easy and versatile cell-based reporter system to identify regulators of cis-SAGe. The reporter, consisting of four main cassettes, simultaneously measures the effects of a candidate regulator on cis-SAGe and canonical splicing. Using this cell-based assay, we screened 102 candidate factors involved in RNA pol II cleavage and termination, elongation, splicing, alternative splicing and R-loop formation. We discovered that two factors, SRRM1 and SF3B1, affect not only cis-SAGe chimeras, but also other types of chimeric RNAs in a genome-wide fashion. This system can be used for studying trans-acting factors and cis-acting sequence elements and factors, as well as for screening small molecule inhibitors.

The iTRAQ-based chloroplast proteomic analysis of Triticum aestivum L. leaves subjected to drought stress and 5-aminolevulinic acid alleviation reveals several proteins involved in the protection of photosynthesis.

BACKGROUNDS: The perturbance of chloroplast proteins is a major cause of photosynthesis inhibition under drought stress. The exogenous application of 5-aminolevulinic acid (ALA) mitigates the damage caused by drought stress, protecting plant growth and development, but the regulatory mechanism behind this process remains obscure. RESULTS: Wheat seedlings were drought treated, and the iTRAQ-based proteomic approach was employed to assess the difference in chloroplast protein content caused by exogenous ALA. A total of 9499 peptides, which could be classified into 2442 protein groups, were identified with ≤0.01 FDR. Moreover, the contents of 87 chloroplast proteins was changed by drought stress alone compared to that of the drought-free control, while the contents of 469 was changed by exogenous ALA application under drought stress compared to that of drought stress alone. The Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results suggested that the ALA pretreatment adjusted some biological pathways, such as metabolic pathways and pathways involved in photosynthesis and ribosomes, to enhance the drought resistance of chloroplasts. Furthermore, the drought-promoted H2O2 accumulation and O2- production in chloroplasts were alleviated by the exogenous pretreatment of ALA, while peroxidase (POD) and glutathione peroxidase (GPX) activities were upregulated, which agreed with the chloroplast proteomic data. We suggested that ALA promoted reactive oxygen species (ROS) scavenging in chloroplasts by regulating enzymatic processes. CONCLUSIONS: Our results from chloroplast proteomics extend the understanding of the mechanisms employed by exogenous ALA to defend against drought stress in wheat.

Dissecting the Cell Entry Pathway of Baculovirus by Single-Particle Tracking and Quantitative Electron Microscopic Analysis.

The budded virus of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infects insect cells through mainly clathrin-mediated endocytosis. However, the cell entry pathway of AcMNPV remains unclear. In this study, by using population-based analysis of single-virus tracking and electron microscopy, we investigated the internalization, fusion behavior, and endocytic trafficking of AcMNPV. AcMNPV internalization into host insect cells was facilitated by actin polymerization and dynamin. After incorporation into early endosomes, the AcMNPV envelope fused with the membranes of early endosome, allowing for nucleocapsid release into the cytoplasm. Microtubules were implicated in the bidirectional and long-range transport of virus-containing endosomes. In addition, microtubule depolymerization reduced the motility of virus-bearing early endosomes, impairing the progression of infection beyond enlarged early endosomes. These findings demonstrated that AcMNPV internalization was facilitated by actin polymerization in a dynamin-dependent manner, and nucleocapsid release occurred in early endosomes in a microtubule-dependent manner. This study provides mechanistic and kinetic insights into AcMNPV infection and enhance our understanding of the infection pathway of baculoviruses.IMPORTANCE Baculoviruses are used widely as environmentally benign pesticides, protein expression systems, and potential mammalian gene delivery vectors. Despite the significant application value, little is known about the cell entry and endocytic trafficking pathways of baculoviruses. In this study, we demonstrated that the alphabaculovirus AcMNPV exhibited actin- and microtubule-dependent transport for nucleocapsid release predominantly from within early endosomes. In contrast to AcMNPV transduction in mammalian cells, its infection in host insect cells is facilitated by actin polymerization for internalization and microtubules for endocytic trafficking within early endosomes, implying that AcMNPV exhibits cell type specificity in the requirement of the cytoskeleton network. In addition, experimental depolymerization of microtubules impaired the progression of infection beyond enlarged early endosomes. This is the first study that dissects the cell entry pathway of baculoviruses in host cells at the single-particle level, which advances our understanding of the early steps of baculovirus entry.

Fusion transcriptome profiling provides insights into alveolar rhabdomyosarcoma.

Gene fusions and fusion products were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of 27 human noncancer tissues, a large number of fusion RNAs were found. By analyzing fusion transcriptome, we observed close clusterings between samples of same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3-FOXO1 (paired box gene 3 fused with forkhead box O1) fusion RNA, which is considered a hallmark of ARMS, was recently found during normal muscle cell differentiation. We performed and analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs. Interestingly, we found that the fusion RNA profile of RH30, an ARMS cell line, is most similar to the myogenesis time point when PAX3-FOXO1 is expressed. In contrast, full transcriptome clustering analysis failed to uncover this connection. Strikingly, all of the 18 chimeric RNAs in RH30 cells could be detected at the same myogenic time point(s). In addition, the seven chimeric RNAs that follow the exact transient expression pattern as PAX3-FOXO1 are specific to rhabdomyosarcoma cells. Further testing with clinical samples also confirmed their specificity to rhabdomyosarcoma. These results provide further support for the link between at least some ARMSs and the PAX3-FOXO1-expressing myogenic cells and demonstrate that fusion RNA profiling can be used to investigate the etiology of fusion-gene-associated cancers.

Linc00210 drives Wnt/ß-catenin signaling activation and liver tumor progression through CTNNBIP1-dependent manner.

BACKGROUND: Liver tumor initiating cells (TICs) have self-renewal and differentiation properties, accounting for tumor initiation, metastasis and drug resistance. Long noncoding RNAs are involved in many physiological and pathological processes, including tumorigenesis. DNA copy number alterations (CNA) participate in tumor formation and progression, while the CNA of lncRNAs and their roles are largely unknown. METHODS: LncRNA CNA was determined by microarray analyses, realtime PCR and DNA FISH. Liver TICs were enriched by surface marker CD133 and oncosphere formation. TIC self-renewal was analyzed by oncosphere formation, tumor initiation and propagation. CRISPRi and ASO were used for lncRNA loss of function. RNA pulldown, western blot and double FISH were used to identify the interaction between lncRNA and CTNNBIP1. RESULTS: Using transcriptome microarray analysis, we identified a frequently amplified long noncoding RNA in liver cancer termed linc00210, which was highly expressed in liver cancer and liver TICs. Linc00210 copy number gain is associated with its high expression in liver cancer and liver TICs. Linc00210 promoted self-renewal and tumor initiating capacity of liver TICs through Wnt/ß-catenin signaling. Linc00210 interacted with CTNNBIP1 and blocked its inhibitory role in Wnt/ß-catenin activation. Linc00210 silencing cells showed enhanced interaction of ß-catenin and CTNNBIP1, and impaired interaction of ß-catenin and TCF/LEF components. We also confirmed linc00210 copy number gain using primary hepatocellular carcinoma (HCC) samples, and found the correlation between linc00210 CNA and Wnt/ß-catenin activation. Of interest, linc00210, CTNNBIP1 and Wnt/ß-catenin signaling targeting can efficiently inhibit tumor growth and progression, and liver TIC propagation. CONCLUSION: With copy-number gain in liver TICs, linc00210 is highly expressed along with liver tumorigenesis. Linc00210 drives the self-renewal and propagation of liver TICs through activating Wnt/ß-catenin signaling. Linc00210 interacts with CTNNBIP1 and blocks the combination between CTNNBIP1 and ß-catenin, driving the activation of Wnt/ß-catenin signaling. Linc00210-CTNNBIP1-Wnt/ß-catenin axis can be targeted for liver TIC elimination.

LncAPC drives Wnt/ß-catenin activation and liver TIC self-renewal through EZH2 mediated APC transcriptional inhibition.

Liver tumor initiating cells (TICs), a small subset cells in tumor bulk, are responsible for liver tumor initiation, metastasis, and relapse. However, the regulatory mechanism of liver TICs remains largely unknown. Here we found a long noncoding RNA lncAPC, locating near from APC locus, was highly expressed in liver cancer and liver TICs. LncAPC was required for liver TIC self-renewal. Silencing and overexpressing lncAPC showed impaired and enhanced sphere formation capacity of liver TICs, respectively. By recruiting EZH2 to APC promoter, LncAPC inhibits APC transcription and thus drives the activation of Wnt/ß-catenin signaling. Attenuate binding between EZH2 and APC promoter was observed upon lncAPC knockdown. What is more, lncAPC-EZH2-APC axis can be targeted to eliminate liver TICs. Altogether, LncAPC promotes liver TIC self-renewal through EZH2-dependent APC transcriptional inhibition.

Recurrent chimeric fusion RNAs in non-cancer tissues and cells.

Gene fusions and their products (RNA and protein) were once thought to be unique features to cancer. However, chimeric RNAs can also be found in normal cells. Here, we performed, curated and analyzed nearly 300 RNA-Seq libraries covering 30 different non-neoplastic human tissues and cells as well as 15 mouse tissues. A large number of fusion transcripts were found. Most fusions were detected only once, while 291 were seen in more than one sample. We focused on the recurrent fusions and performed RNA and protein level validations on a subset. We characterized these fusions based on various features of the fusions, and their parental genes. They tend to be expressed at higher levels relative to their parental genes than the non-recurrent ones. Over half of the recurrent fusions involve neighboring genes transcribing in the same direction. A few sequence motifs were found enriched close to the fusion junction sites. We performed functional analyses on a few widely expressed fusions, and found that silencing them resulted in dramatic reduction in normal cell growth and/or motility. Most chimeras use canonical splicing sites, thus are likely products of 'intergenic splicing'. We also explored the implications of these non-pathological fusions in cancer and in evolution.

Discovery of CTCF-sensitive Cis-spliced fusion RNAs between adjacent genes in human prostate cells.

Genes or their encoded products are not expected to mingle with each other unless in some disease situations. In cancer, a frequent mechanism that can produce gene fusions is chromosomal rearrangement. However, recent discoveries of RNA trans-splicing and cis-splicing between adjacent genes (cis-SAGe) support for other mechanisms in generating fusion RNAs. In our transcriptome analyses of 28 prostate normal and cancer samples, 30% fusion RNAs on average are the transcripts that contain exons belonging to same-strand neighboring genes. These fusion RNAs may be the products of cis-SAGe, which was previously thought to be rare. To validate this finding and to better understand the phenomenon, we used LNCaP, a prostate cell line as a model, and identified 16 additional cis-SAGe events by silencing transcription factor CTCF and paired-end RNA sequencing. About half of the fusions are expressed at a significant level compared to their parental genes. Silencing one of the in-frame fusions resulted in reduced cell motility. Most out-of-frame fusions are likely to function as non-coding RNAs. The majority of the 16 fusions are also detected in other prostate cell lines, as well as in the 14 clinical prostate normal and cancer pairs. By studying the features associated with these fusions, we developed a set of rules: 1) the parental genes are same-strand-neighboring genes; 2) the distance between the genes is within 30kb; 3) the 5' genes are actively transcribing; and 4) the chimeras tend to have the second-to-last exon in the 5' genes joined to the second exon in the 3' genes. We then randomly selected 20 neighboring genes in the genome, and detected four fusion events using these rules in prostate cancer and non-cancerous cells. These results suggest that splicing between neighboring gene transcripts is a rather frequent phenomenon, and it is not a feature unique to cancer cells.

A protein-encoding CCDC7 circular RNA inhibits the progression of prostate cancer by up-regulating FLRT3.

Circular RNAs (circRNAs) are a family of endogenous RNAs that have become a focus of biological research in recent years. Emerging evidence has revealed that circRNAs exert biological functions by acting as transcriptional regulators, microRNA sponges, and binding partners with RNA-binding proteins. However, few studies have identified coding circRNAs, which may lead to a hidden repertoire of proteins. In this study, we unexpectedly discovered a protein-encoding circular RNA circCCDC7(15,16,17,18,19) while we were searching for prostate cancer related chimeric RNAs. circCCDC7(15,16,17,18,19) is derived from exon 19 back spliced to exon 15 of the CCDC7 gene. It is significantly downregulated in patients with high Gleason score. Prostate cancer patients with decreased circCCDC7(15,16,17,18,19) expression have a worse prognosis, while linear CCDC7 had no such association. Overexpressed circCCDC7(15,16,17,18,19) inhibited prostate cancer cell migration, invasion, and viability, supporting classification of circCCDC7(15,16,17,18,19) as a bona fide tumor suppressor gene. We provide evidence that its tumor suppressive activity is driven by the protein it encodes, and that circCCDC7(15,16,17,18,19) encodes a secretory protein. Consistently, conditioned media from circCCDC7(15,16,17,18,19) overexpressing cells has the same tumor suppressive activity. We further demonstrate that the tumor suppressive activity of circCCDC7(15,16,17,18,19) is at least partially mediated by FLRT3, whose expression also negatively correlates with Gleason score and clinical prognosis. In conclusion, circCCDC7(15,16,17,18,19) functions as a tumor suppressor in prostate cancer cells through the circCCDC7-180aa secretory protein it encodes, and is a promising therapeutic peptide for prostate cancer.

UBA1-CDK16 : A Sex-Specific Chimeric RNA and Its Role in Immune Sexual Dimorphism.

RNA processing mechanisms, such as alternative splicing and RNA editing, have been recognized as critical means to expand the transcriptome. Chimeric RNAs formed by intergenic splicing provide another potential layer of RNA diversification. By analyzing a large set of RNA-Seq data and validating results in over 1,200 blood samples, we identified UBA1-CDK16 , a female-specific chimeric transcript. Intriguingly, both parental genes, are expressed in males and females. Mechanistically, UBA1-CDK16 is produced by cis-splicing between the two adjacent X-linked genes, originating from the inactive X chromosome. A female-specific chromatin loop, formed between the junction sites, facilitates the alternative splicing of its readthrough precursor. This unique chimeric transcript exhibits evolutionary conservation, evolving to be female-specific from non-human primates to humans. Furthermore, our investigation reveals that UBA1-CDK16 is enriched in the myeloid lineage and plays a regulatory role in myeloid differentiation. Notably, female COVID-19 patients who tested negative for this chimeric transcript displayed higher counts of neutrophils, highlighting its potential role in disease pathogenesis. These findings support the notion that chimeric RNAs represent a new repertoire of transcripts that can be regulated independently from the parental genes, and a new class of RNA variance with potential implications in sexual dimorphism and immune responses.

CTNNBIP1-CLSTN1 Functions as a Housekeeping Chimeric RNA, and Regulates Cell Proliferation through SERPINE2.

The conventional wisdom that chimeric RNAs being peculiarity of carcinoma, and the products of chromosomal rearrangement is being challenged, However, experimental evidence supporting chimeric RNAs in normal physiology being functional is scarce. We decided to focus on one particular chimeric RNA, CTNNBIP1-CLSTN1 . We examined its expression among various tissues and cell types, and compared quantitatively among cancer and non-cancer cells. We further investigated its role in a panel of non-cancer cells and probed the functional mechanism. We found that this fusion transcript is expressed in almost all tissues, and a wide range of cell types including fibroblasts, epithelial, stem, vascular endothelial cells, and hepatocytes. The expression level in non-cancerous cell lines is also not evidently different from that in the cancer cell lines. Furthermore, silencing CTNNBIP1-CLSTN1 significantly reduces cell proliferation rate, by inducing G2/M arrest in cell cycle progress and apoptosis in at least three cell types. Importantly, rescue experiments confirmed that the cell cycle arrest can be regained by exogenous expression of the chimera, but not the wild type parental gene. Further evidence is provided that CTNNBIP1-CLSTN1 regulates cell proliferation through SERPINE2 . Thus, CTNNBIP1-CLSTN1 represents an example of a new class of fusion RNA, dubbed "housekeeping chimeric RNAs".

CTNNBIP1-CLSTN1 functions as a housekeeping chimeric RNA and regulates cell proliferation through SERPINE2.

The conventional understanding that chimeric RNAs are unique to carcinoma and are the products of chromosomal rearrangement is being challenged. However, experimental evidence supporting the function of chimeric RNAs in normal physiology is scarce. We decided to focus on one particular chimeric RNA, CTNNBIP1-CLSTN1. We examined its expression in various tissues and cell types and compared it quantitatively among cancer and noncancer cells. We further investigated its role in a panel of noncancer cells and investigated the functional mechanism. We found that this fusion transcript is expressed in almost all tissues and a wide range of cell types, including fibroblasts, epithelial cells, stem cells, vascular endothelial cells, and hepatocytes. In addition, the CTNNBIP1-CLSTN1 expression level in noncancerous cell lines was not evidently different from that in cancer cell lines. Furthermore, in at least three cell types, silencing CTNNBIP1-CLSTN1 significantly reduced the cell proliferation rate by inducing G2/M arrest and apoptosis. Importantly, rescue experiments confirmed that cell cycle arrest was restored by exogenous expression of the chimera but not the wild-type parental gene. Further evidence is provided that CTNNBIP1-CLSTN1 regulates cell proliferation through SERPINE2. Thus, CTNNBIP1-CLSTN1 is an example of a new class of fusion RNAs, dubbed "housekeeping chimeric RNAs".

Draft genome sequence of Rhodococcus sp. strain P14, a biodegrader of high-molecular-weight polycyclic aromatic hydrocarbons.

The genus Rhodococcus is known for its ability to degrade various xenobiotic compounds. Rhodococcus sp. strain P14 isolated from crude oil-contaminated sediments can degrade mineral oil and polycyclic aromatic hydrocarbons (PAHs). The draft genome sequence of Rhodococcus sp. P14 was obtained using Solexa technology, which provided an invaluable genetic background for further investigation of the ability of P14 to degrade xenobiotic compounds.

Characterization and Function of Glycans on the Spike Proteins of SARS-CoV-2 Variants of Concern.

SARS-CoV-2 variants of concern (VOCs) pose a great challenge to viral prevention and treatment owing to spike (S) protein mutations, which enhance their infectivity and capacity for immune evasion. However, whether these S protein mutations affect glycosylation patterns and thereby influence infectivity and immunogenicity remains unclear. In this study, four VOC S proteins-S-Alpha, S-Beta, S-Delta, and S-Omicron-were expressed and purified. Lectin microarrays were performed to characterize their glycosylation patterns. Several glycans were differentially expressed among the four VOC S proteins. Furthermore, the functional examination of glycans differentially expressed on S-Omicron revealed a higher expression of fucose-containing glycans, which modestly increased the binding of S-Omicron to angiotensin converting enzyme 2 (ACE2). A higher abundance of sialic acid and galactose-containing glycan was observed on S-Omicron, which significantly reduced its sensitivity against broad S protein-neutralizing antibodies. These findings contribute to the further understanding of SARS-CoV-2 infection mechanisms and provide novel glycan targets for emerging and future variants of SARS-CoV-2. IMPORTANCE Though glycosylation sites of SARS-CoV-2 S protein remain highly conserved, we confirmed that mutations in the Spike gene affect the S protein glycan expression pattern in different variants. More importantly, we found that glycans were differentially expressed on the S protein of the Omicron variant, enabling different forms of receptor binding and neutralization resistance. This study improves our understanding of SARS-CoV-2 glycomics and glycobiology and provides novel therapeutic and preventive strategies for SARS-CoV-2 VOCs.

Profile of chimeric RNAs and TMPRSS2-ERG e2e4 isoform in neuroendocrine prostate cancer.

PURPOSE: Specific gene fusions and their fusion products (chimeric RNA and protein) have served as ideal diagnostic markers and therapeutic targets for cancer. However, few systematic studies for chimeric RNAs have been conducted in neuroendocrine prostate cancer (NEPC). In this study, we explored the landscape of chimeric RNAs in different types of prostate cancer (PCa) cell lines and aimed to identify chimeric RNAs specifically expressed in NEPC. METHODS: To do so, we employed the RNA-seq data of eight prostate related cell lines from Cancer Cell Line Encyclopedia (CCLE) for chimeric RNA identification. Multiple filtering criteria were used and the candidate chimeric RNAs were characterized at multiple levels and from various angles. We then performed experimental validation on all 80 candidates, and focused on the ones that are specific to NEPC. Lastly, we studied the clinical relevance and effect of one chimera in neuroendocrine process. RESULTS: Out of 80 candidates, 15 were confirmed to be expressed preferentially in NEPC lines. Among them, 13 of the 15 were found to be specifically expressed in NEPC, and four were further validated in another NEPC cell line. Importantly, in silico analysis showed that tumor malignancy may be correlated to the level of these chimeric RNAs. Clinically, the expression of TMPRSS2-ERG (e2e4) was elevated in tumor tissues and indicated poor clinical prognosis, whereas the parental wild type transcripts had no such association. Furthermore, compared to the most frequently detected TMPRSS2-ERG form (e1e4), e2e4 encodes 31 more amino acids and accelerated neuroendocrine process of prostate cancer. CONCLUSIONS: In summary, these findings painted the landscape of chimeric RNA in NEPC and supported the idea that some chimeric RNAs may represent additional biomarkers and/or treatment targets independent of parental gene transcripts.

Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes.

Infantile (fetal and neonatal) megakaryocytes (Mks) have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell-derived Mks. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with the potential to affect MKL1 function and found that DYRK1A inhibition dramatically enhanced Mk morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal Mks. Mks derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss-of-function studies confirmed MKL1 involvement in this morphogenetic pathway. Expression of Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile Mks.