DDX24, a D-E-A-D box RNA helicase, is required for muscle fiber organization and anterior pole specification essential for head regeneration in planarians

Planarians have a remarkable ability to undergo whole-body regeneration. The timely establishment of polarity at the wound site followed by the specification of the organizing centers- the anterior pole and the posterior pole, are indispensable for successful regeneration. In planarians, polarity, pole, and positional-information determinants are predominantly expressed by muscles. The molecular toolkit that enables this functionality of planarian muscles however remains poorly understood. Here we report that SMED_DDX24, a D-E-A-D Box RNA helicase and the homolog of human DDX24, is critical for planarian head regeneration. DDX24 is enriched in muscles and its knockdown leads to defective muscle-fiber organization and failure to re-specify anterior pole/organizer. Overall, loss of DDX24 manifests into gross misregulation of many well-characterized positional-control genes and patterning-control genes, necessary for organogenesis and tissue positioning and tissue patterning. In addition, wound-induced Wnt signalling was also upregulated in ddx24 RNAi animals. Canonical WNT-βCATENIN signalling is known to suppress head identity throughout bilateria, including planarians. Modulating this Wnt activity by β-catenin-1 RNAi, the effector molecule of this pathway, partially rescues the ddx24 RNAi phenotype, implying that a high Wnt environment in ddx24 knockdown animals likely impedes their normal head regeneration. Furthermore, at a sub-cellular level, RNA helicases are known to regulate muscle mass and function by regulating their translational landscape. ddx24 knockdown leads to the downregulation of large subunit ribosomal RNA and the 80S ribosome peak, implying its role in ribosome biogenesis and thereby influencing the translational output. This aspect seems to be an evolutionarily conserved role of DDX24. In summary, our work demonstrates the role of a D-E-A-D box RNA helicase in whole-body regeneration through muscle fiber organization, and pole and positional-information re-specification, likely mediated through translation regulation.

ABSTRACT: Planarians have a remarkable ability to undergo whole-body regeneration. The 21 timely establishment of polarity at the wound site followed by the specification of the 22 organizing centers-the anterior pole and the posterior pole, are indispensable for successful 23 regeneration. In planarians, polarity, pole, and positional-information determinants are 24 predominantly expressed by muscles. The molecular toolkit that enables this functionality of 25 planarian muscles however remains poorly understood. Here we report that SMED_DDX24, a 26 D-E-A-D Box RNA helicase and the homolog of human DDX24, is critical for planarian head 27 regeneration. DDX24 is enriched in muscles and its knockdown leads to defective muscle-fiber 28 organization and failure to re-specify anterior pole/organizer. Overall, loss of DDX24 29 manifests into gross misregulation of many well-characterized positional-control genes and 30 patterning-control genes, necessary for organogenesis and tissue positioning and tissue 31 patterning. In addition, wound-induced Wnt signalling was also upregulated in ddx24 RNAi 32 animals. Canonical WNT-βCATENIN signalling is known to suppress head identity 33 throughout bilateria, including planarians. Modulating this Wnt activity by β-catenin-1 RNAi, 34 the effector molecule of this pathway, partially rescues the ddx24 RNAi phenotype, implying 35 that a high Wnt environment in ddx24 knockdown animals likely impedes their normal head 36 regeneration. Furthermore, at a sub-cellular level, RNA helicases are known to regulate muscle 37 mass and function by regulating their translational landscape. ddx24 knockdown leads to the 38 downregulation of large subunit ribosomal RNA and the 80S ribosome peak, implying its role 39 in ribosome biogenesis and thereby influencing the translational output. This aspect seems to INTRODUCTION required for regulating Wnt activity essential for proper localization of signaling centers 95 essential for regeneration. 96 Like many other tissues, muscle function was shown to be critically dependent on its 97 translational landscape, which in turn is primarily controlled by regulating ribosome biogenesis 98 (42,(45)(46)(47)(48)(49). Knockdown of ddx24 in planarians resulted in reduced levels of large subunit 99 ribosomal RNA (28S rRNA) as well as reduced 80S ribosome peak. This aspect appears to be 100 an evolutionarily conserved role of DDX24 (50,51). Since DDX24 protein was enriched in 101 muscles, these results suggested that DDX24 could enable the functional state of these muscles 102 by regulating their translational output via ribosome biogenesis.   Immunostaining with anti-arrestin antibody and RNA-FISH for opsin, which cumulatively 118 marks the PRN, optic chaisma, and cell body of the PRN (52,53), showed defective photoreceptor organization in the ddx24 KD animals (Figure 1b). We then sought to investigate 120 the status of other organ systems in our knockdown animals. In planarians, the cephalic ganglia, 121 i.e., its brain, is a bilobed structure (54), which can be visualized by the staining with nuclear 122 dyes such as Hoechst or by immunostaining with anti-Gα q/11/14 antibody. In ddx24 RNAi 123 animals, the planarian brain either failed to regenerate completely, or a rudimentary brain was 124 formed compared to the control animals ( Figure 1c). 125 Planarian flatworms belong to the order tricladida characterized by the presence of three 126 primary gut branches-one anterior primary gut branch that splits pre-pharyngeally into two 127 posterior primary gut branches (55). dsRNA treated animals were amputated post-pharyngeally 128 such that the posterior gut branches merge and then subsequently regenerate the entire anterior 129 gut branch (Figure 1-figure supplement 1b). RNA in-situ for mat, a gut marker (56), clearly 130 showed that ddx24 KD animals failed to regenerate the anterior branch of the intestine ( Figure   131 1d). Also, no pharyngeal cavity (marked by Asterix *) was observed in these knockdown  Together, our data shows that loss of DDX24 completely impaired anterior regeneration.   of piwi-1+ cells, a pan-neoblast marker, also expressed ddx24 (Figure 3c). Since we found 171 DDX24 protein in muscles and stem cells, we hypothesized that a fraction of the neoblasts that 172 expressed the ddx24 could be a neoblast population primed for muscle lineage. To test this, 173 we performed double-RNA-FISH using in situ probes for ddx24 and collagen, in combination 174 with immunostaining using an antibody against PIWI-1 protein, a marker for pluripotent 175 neoblasts and early progenitors (i.e., immediate neoblast progeny) (60). Our results showed 176 that 36.5 ± 9.7% of ddx24-PIWI1 double-positive cells were also positive for collagen ( Figure   177 3d). This suggested that a subset of ddx24+ neoblasts were indeed primed for muscle fate. This  It is worth mentioning that even though single-cell RNA-seq datasets point towards the 182 expression of ddx24 transcript in other lineages, like the epidermis, gut, neural, and pharynx, 183 our antibody did not detect DDX24 protein in any of these differentiated planarian tissues.

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However, in intact animals, DDX24 expression was detected in cells other than muscle fibers 185 whose identity we were unable to determine (    (fold-change (ddx24 KD/Control KD) = 3.14; adjusted p-value = 0.0009). Therefore, it is likely that the activity of canonical WNT-βCATENIN signaling is upregulated in absence of DDX24 270 and this could be one potential reason why ddx24 KD planarians fail to regenerate their head.

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To further test this idea, we performed a double knockdown of β-catenin-1 and ddx24. We  ddx24 KD leads to downregulation of ribosomal RNA processing machinery 284 We next sought to uncover one potential sub-cellular mechanism how DDX24, an RNA 285 helicase, could affect muscle architecture and function, thereby affecting whole-body 286 regeneration. GO analysis of our bulk RNA sequencing data revealed that the expression of 287 many genes associated with ribosomal RNA processing machinery was de-regulated in ddx24 288 KD animals ( Figure 6a). Mak5, the yeast homolog of DDX24, was shown to be essential for 289 large subunit ribosomal RNA maturation (50,51,74). In planarians, the qRT-PCR analysis 290 revealed ~40% downregulation in 28S rRNA levels upon ddx24 KD whereas 18S rRNA levels 291 remain nearly unchanged ( Figure 6b). Therefore, our data hinted towards potential mis-292 regulation in translation mediated by reduced levels of mature 80S ribosomal. This was tested 293 by performing polysome profiling on planarian lysates from control and knockdown animals.
As predicted, the 80S ribosome peak was reduced in the absence of DDX24, which is 295 suggestive of defective ribosome assembly leading to a perturbed translational landscape 296 ( Figure 6c). Since DDX24 protein was enriched in body wall muscles, and a subset of X1 297 neoblasts, we speculated that defective ribosome biogenesis in ddx24 KD animals was 298 predominantly restricted to these specific compartments. Therefore, it is likely that muscle 299 defects in ddx24 KD animals, both in terms of muscle fiber organization as-well-as failure to 300 specify anterior-pole cells, could be a result of defective translational machinery in these cells.

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In conclusion, our work highlights the role of translation regulation mediated by a D-E-A-D 302 Box RNA helicase in whole-body regeneration via maintaining muscle form and function.  We conducted an RNAi screen for different post-transcriptional gene regulators, including 339 members from the D-E-A-D box RNA helicase family, in Schmidtea mediterranea, the go-to 340 model system to study whole-body regeneration. Here, we report that SMED_DDX24, the 341 Schmidtea mediterranea homolog of human DDX24, is essential for its regeneration. Although 342 DDX24 is conserved across eukaryotes, little is known about the function of this protein in 343 regulating various biological processes. In this study, we have mostly focused our effort to understand how DDX24 governs head regeneration in planarians. We found that ddx24 mRNA 345 is expressed by a population of neoblasts, neoblasts primed for muscle fate, and differentiated 346 muscles. However, the protein is highly enriched in body-wall muscle fiber, particularly 347 diagonal and longitudinal muscle fibers. We also detected the protein in a subset of neoblasts.

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Although, the loss of DDX24 did not affect piwi1+ neoblasts, their maintenance, proliferation, 349 or their overt differentiation. However, the loss of DDX24 did severely affect muscle fiber is likely that DDX24 is required for organizing muscle fibers and not for their overall fate 355 determination. 356 We also found the co-expression of ddx24 with the anterior pole markers, both in intact and in     Olympus FV 3000 laser scanning microscope. Images were processed using Fiji.

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(https://imagej.net/Fiji). All cell counting was performed manually. and was loaded onto a sucrose gradient (15% to 45%) and centrifuged at 40,000 RPM for 2 to 503 2.5 hours at 4°C. The sucrose gradient was then loaded onto an ISCO Teledyne UA-6 504 Absorbance Detector. Sixty percent sucrose was pumped from below to push the sucrose 505 gradient to the UV chamber. The sensitivity parameter of the machine was set at 0.5. were sequenced for every sample. These reads were adapter trimmed using Trimmomatic (95) and mapped to rRNA and other contamination databases. Reads that did not align with these 514 databases were taken for further analysis. We used reference-based transcriptome assembly