Post-transcriptional regulation in planarian stem cells

G Model

ARTICLE IN PRESS

YSCDB-2586; No. of Pages 10

Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb

Review

Post-transcriptional regulation in planarian stem cells Srikar Krishna a,b , Dasaradhi Palakodeti b , Jordi Solana c,∗ a

SASTRA University, Thanjavur, Tamil Nadu, India Institute for Stem Cell Biology and Regenerative Medicine, GKVK Campus, Bellary Road, Bengaluru, India c Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford, UK b

a r t i c l e

i n f o

Article history: Received 26 January 2018 Received in revised form 11 May 2018 Accepted 14 May 2018 Available online xxx Keywords: Planarian Neoblast Stem cell Post-transcriptional regulation RNA binding protein

a b s t r a c t Planarians are known for their immense regenerative abilities. A pluripotent stem cell population provides the cellular source for this process, as well as for the homeostatic cell turnover of the animals. These stem cells, known as neoblasts, present striking similarities at the morphological and molecular level to germ cells, but however, give rise to somatic tissue. Many RNA binding proteins known to be important for germ cell biology are also required for neoblast function, highlighting the importance of post-transcriptional regulation for stem cell control. Many of its aspects, including alternative splicing, alternative polyadenylation, translational control and mRNA deadenylation, as well as small RNAs such as microRNAs and piRNA are critical for stem cells. Their inhibition often abrogates both regeneration and cell turnover, resulting in lethality. Some of aspects of post-transcriptional regulation are conserved from planarian to mammalian stem cells. © 2018 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Many post-transcriptional regulators are highly expressed and are key regulators of planarian stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Stem cell chromatoid bodies and the germline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The life of an mRNA: post-transcriptional gene regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The classic germline RBPs in planarian stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Inside of the nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.1. The regulation of alternative splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.2. The regulation of alternative polyadenylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Outside of the nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 7.1. Translational control and mRNA degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Small RNAs in planarian stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction Freshwater planarians are well known for their remarkable regenerative capacities by virtue of an abundant pool of pluripotent stem cells called the neoblasts [1–3]. They are not only the source of

Abbreviations: RBP, RNA binding protein; GMP, germline multipotency program; PriSCs, primordial stem cells; UTR, untranslated region; ApA, alternative polyadenylation; FACS, Fluorescence Activated Cell Sorting; PAS, Polyadenylation Signal. ∗ Corresponding author. E-mail address: [email protected] (J. Solana).

regenerative cells but sustain the cell turnover of the animal. Consequently, eliminating all stem cells by means of irradiation or by genetic means results in an immediate loss of regenerative power but also in interrupted cell turnover. Planarians are able to survive with no observable defects for a few days, but after the first week their heads regress and their bodies curl. These are the phenotypic consequences of interrupted cell turnover, with some differentiated cell types becoming scarce. The process results in lethality. However, both regeneration and cell turnover defects can be rescued by injecting single neoblasts [4]. This landmark study showed

https://doi.org/10.1016/j.semcdb.2018.05.013 1084-9521/© 2018 Elsevier Ltd. All rights reserved.

Please cite this article in press as: S. Krishna, et al., Post-transcriptional regulation in planarian stem cells, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.013

G Model

ARTICLE IN PRESS

YSCDB-2586; No. of Pages 10

S. Krishna et al. / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

2

Table 1 Post-transcriptional regulatory genes characterized in planaria. Gene

Type and Function

Expression in planaria

Phenotype

Smed-Vasa1

RNA Helicase

Clonogenic Neoblasts

Smedwi-1 Smedwi-2

Piwi Protein; piRNA pathway Piwi Protein; piRNA pathway

Neoblasts Neoblasts

Smedwi-3

Piwi Protein; piRNA pathway

Neoblasts

Smed-Ago2

miRNA pathway; mRNA degradation and translational regulation CELF family of RNA binding proteins; Alternative Splicing

Neoblasts and differentiated cells

Lesions on Dorsal Epidermis; Failure in neoblast proliferation No robust phenotypes observed Regeneration defects; Ventral curling; Head regression; Failure in neoblast proliferation Regeneration defects; Ventral curling; Head regression; Failure in neoblast proliferation Head regression; Lysis; Severe regeneration defects

Smed-bruli

Spoltud-1

Tudor domain containing protein

Djpum

PUF RNA-binding protein

Smed-mbnl-1 Smed-pabpc1

Smed-nanos

MBNL Protein; Alternative splicing PolyA binding protein; mRNA stability; Translation PolyA binding protein; mRNA stability; Translation Zinc finger protein

Smed-not1

mRNA deadenylation

Smed-mex3-1

KF domain containing RNA binding protein

Smed-pabpc2

Neoblasts; Cephalic ganglia; Ventral nerve cord Neurons; Germ cells and Neoblasts (chromatoid bodies) Cephalic ganglia; Neoblasts Differentiated cells and progenitors Germline Neoblasts; Epidermal lineage; Gut Mature testes and Ovaries; Germline primordia in asexuals; Eye precursors Ubiquitous Epithelial progeny and Neoblasts

that planarian stem cells are pluripotent: one stem cell can give rise to all differentiated cell types of a planarian. Planarians are a very convenient model to study stem cells and their regulatory mechanisms. Planarian stem cells are constantly differentiating to all mature cell types, and this enables the process to be studied in vivo rather than in vitro. Neoblasts are easy to localise by labelling techniques (by in situ hybridisation or immunolocalisation of Smedwi-1) [5], easy to isolate in large numbers by Fluorescence Activated Cell Sorting (FACS) [6], easy to eliminate by irradiation or genetic means and easy to functionally study by RNAi [7]. All of these techniques, in combination with genomic [8,9] and transcriptomic [10–12] knowledge has spurred a myriad of studies regarding their biology. We have a very good picture of their transcriptomic profiles by RNAsequencing [13–17] and we are beginning to understand their differentiation process at the single-cell level [18–23]. These studies uncovered the expression of many transcriptional, epigenetic and post-transcriptional regulators in planarian stem cells. Here, we will focus on post-transcriptional regulation, a process shown to be critical for neoblast function and stem cell biology. 2. Many post-transcriptional regulators are highly expressed and are key regulators of planarian stem cells After RNA-sequencing technologies became available, several groups profiled the planarian stem cell transcriptome. Neoblasts were either sorted out by FACS and compared to differentiated cells [13,15,16] or eliminated by RNAi and compared to control planarians [17]. Several of these studies highlighted the presence of RNA-binding proteins (RBPs) and post-transcriptional regulators as top markers of planarian neoblasts. However, the planarian community had already established the importance of RBPs in several landmark studies involving planarian neoblast. Originally, Shibata and co-workers identified the gene expression of vasarelated genes in planarian neoblasts [24]. Several studies followed

Regeneration defects; Ventral curling; Head regression; Failure in neoblast proliferation Delayed regeneration; Regeneration defects Failure in blastema formation; Regeneration defects and Lysis Mild regeneration defects Absence of spermatids in the testes Failure in regeneration; Lesions and Lysis Not known

Regeneration defects; Ventral curling; Head regression Ventral curling; head regression and lysis; Regeneration defects

and found homologues of other classic RNA-binding proteins such as Pumilio [25], Piwi [5], Tudor [26] and Bruno [27] expressed in planarian neoblasts. Knockdown of many of these factors also abrogated regeneration and affected neoblasts at different levels (Table 1). 3. Stem cell chromatoid bodies and the germline A number of studies published in the last decade have theorized that planarian stem cells resemble the germline cells of other organisms. This notion was brought about by the presence of large electron-dense granules -called chromatoid bodies- in planarian neoblasts that resemble those that had been observed in the germ cells of almost every animal. The germ cells typically express genes such as Vasa, Pumilio, Piwi, Tudor and Bruno, and so do planarian neoblasts [28]. These observations strongly suggested a connection between neoblasts and germ cells that is substantiated by the expression of the same RBPs. The chromatoid bodies are perinuclear electron-dense granules [29], devoid of any membrane. They are often found in close association with nuclear pores as well as mitochondria. Chromatoid bodies resemble the germ granules [30,31], often called nuage, present in germ cells of almost every organism examined. Indeed, they are also found in planarian germline stem cells [26]. While only a few proteins have been shown to localise to planarian chromatoid bodies [26,32,33], this is in part due to a lack of antibodies that are specific to the planarian chromatoid body constituents. However, there is little doubt that planarian chromatoid bodies are similar in composition to germ granules. Thus, planarian stem cells resemble germline cells both by the presence of RNA granules and by the expression of well-known germline markers. The significance of this is still enigmatic and a subject of debate. In fact, planarian neoblasts are not the only stem cell with somatic potential that has these characteristics. Stem cells from sponges, cnidarians and acoels, for instance, also express

Please cite this article in press as: S. Krishna, et al., Post-transcriptional regulation in planarian stem cells, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.013

G Model YSCDB-2586; No. of Pages 10

ARTICLE IN PRESS S. Krishna et al. / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

3

germline markers. Embryonic populations of pluripotent or multipotent cells from animals as diverse as annelids [34] or sea urchins [35] also express germline markers. This has led to the proposal of a germline multipotency program (GMP, comprising germline markers such as Vasa, Nanos and Piwi) that is often found not only in germline cells but in multipotent or pluripotent stem cells that can give rise to somatic tissues [36]. Altogether this also led to the proposal that these cells are an integral part of the germline cycle called the Primordial Stem Cells [28]. According to the model these cells can sometimes self-renew until adulthood. Often this is seen in animals with high regenerative powers. In these animals, the Primordial Stem Cell (PriSC) populations in the adult are capable of transmitting the genome from generation to generation via asexual reproduction or germline regeneration. This shared function with the sexual germline can explain their resemblances at the cellular and molecular level.

The logic and syntax of RBP mediated regulation is still vastly unknown. The interplay between mRNAs and RBPs is quite complex. While most mRNAs exhibit binding sites for different RBPs, several RBPs can also bind to different mRNAs. Further, many RBPs possess different types of RNA-binding domains, thus intensifying the complexity in RBP mediated regulation. It is fair to assume that this regulation is exerted in a highly combinatorial way. The site of binding can also confer specificity of regulation. For instance, in the context of alternative splicing, RBPs such as Nova and MBNL are associated with exon inclusion when binding downstream of the exon, but exon exclusion when binding in the exon or upstream of it [45]. A special case of this are the Argonaute family of RBPs: their specificity is conferred by the loading of a small RNA that directs binding to a complementary RNA sequence. Some other RBPs do not have any binding specificity and bind generally or are targeted by other proteins.

4. The life of an mRNA: post-transcriptional gene regulation

5. The classic germline RBPs in planarian stem cells

Post-transcriptional regulation is therefore important for stem cells from many organisms and germ cells in general, and for planarian stem cells in particular. Most proteins involved in posttranscriptional regulation are RNA binding proteins like the main germ and stem cell markers. The human genome encodes 1542 RBPs [37], similar to the number of transcription factors. We annotated more than 300 RBPs in the planarian transcriptome [38], but there are likely many more. Among them are members of most known RBP families, including RNA-helicases, KH, ZnF, CSD and RRM domain proteins. mRNAs are synthesized in the nucleus (Fig. 1). Epigenetic regulation of chromatin makes the genomic regions that encode an mRNA accessible and transcriptionally active. Transcriptional regulation brings the right transcriptional complexes. RNA polymerase II then transcribes a precursor mRNA (pre-mRNA). From this step on, post-transcriptional gene regulation assumes control [39]. The first step is mRNA processing: pre-mRNAs need to be capped, spliced, cleaved at their 3 ends and polyadenylated. All of these processes can be subject to context-specific regulation. For instance, alternative splicing leads to different mRNA isoforms that contain different sets of exons, intron retention leads to the inclusion of an intronic sequence in the mature mRNA and alternative cleavage and polyadenylation leads to the production of mRNA isoforms of different 3 UTR length. Several mRNA quality control mechanisms also take place after synthesis. mRNAs then need to be exported out of the nucleus. In the cytoplasm, mRNAs can undergo several processes. Some mRNAs are targeted to different subcellular localisations. Some other mRNAs can be translationally repressed. The ultimate goal of an mRNA is to be translated by ribosomes. Finally, mRNAs are degraded in the cytoplasm. The first step of mRNA degradation is deadenylation. Context specific regulation of these processes leads to alternative mRNA localisation, translational repression or selective degradation. mRNAs are bound by RBPs throughout their lifetime, especially in their 3 UTRs. These interactions mediate the different steps of mRNA maturation and target mRNAs to large cellular complexes such as the spliceosome or the ribosome. RBPs directly bind mRNA via RNA binding domains [40]. The most common RNA binding domains are the RNA Recognition Motif (RRM), the K homology (KH) domain, the double-stranded RNA-binding domain (dsRBD) and the zinc finger (ZnF) domain. Some of these exclusively bind RNA while other, such as the ZnF motifs, can be RNA binding or DNA binding. RNA-protein interactions can be sequence-specific. Motifs in the RNA sequence are recognised by the RNA-binding domain. These binding motifs can be studied by techniques such as CLIP [41–43] and RNA-compete [44].

Vasa is a DEAD-box RNA helicase that is found to be expressed in the germline of almost every animal studied [46]. RNA helicases are proteins that can unwind long double stranded regions or short local secondary structures [47]. They have been involved in almost every step of RNA metabolism. Vasa, an orthologue of the human DDX4, is a translational activator that is localised to the nuage granules in Drosophila melanogaster. Vasa homologues have been found as well in pluripotent cells from different organisms, such as those of the annelid Mesodermal Posterior Growth Zone [34] or the small micromere lineage of sea urchins [35]. Vasa is therefore one of the key components of the GMP and a key marker of the PriSCs. Vasarelated genes were very early found to be expressed in planarian neoblasts [24]. These genes seem to belong to the DDX3/PL10/Belle group of homology [48], an RNA helicase that is very closely related to Vasa. Later, two Vasa homologues were described in Schmidtea mediterranea [49]. Smed-vasa-1 was found to be needed for neoblast clonogenic expansion and neoblast differentiation (Table 1). The fly tudor germline gene encodes for a large protein with many Tudor domains. Other Tudor proteins of other organisms are made of a smaller number of Tudor domains, and sometimes in combination with other, often RNA-binding, domains. Although the Tudor domain is not an RNA-binding domain itself, Tudor domains are involved in several aspects of post-transcriptional regulation. The Tudor domain has high affinity for methylated arginine or lysine residues [50]. These are present in many proteins, including histones and Piwi proteins. Tudor domains are believed to act as readers of these marks, bringing other effector proteins. There are at least 10 Tudor domain containing proteins in Schmidtea mediterranea, and other species have a similar number of homologues. The Schmidtea polychroa Spoltud-1 gene [26] codes for a protein with 3 Tudor domains and an RRM domain, and thus is likely an RNA binding protein. The SPOLTUD-1 protein localises to chromatoid bodies in neoblasts, as well as RNA granules present in neurons and germ cells. Long term inhibition of Spoltud-1 results in neoblast loss and lack of regenerative capacity (Table 1). The mechanisms by which Spoltud-1 function, or its interaction patterns are still unknown. The RBP Nanos is a special case of GMP gene in planarians: it is normally expressed both in germ cells and stem cells from different organisms [36] but it is exclusive to the germ cells of planarians [51,52]. It is essential for the development and regeneration of the germ cells in S. mediterranea. In juvenile and asexual worms nanos transcripts are present in a subset of neoblast-like cells with a distribution similar to that of the testis of sexual animals that are assumed to be the Primordial Germ Cells of the animal. Pumilio is yet another RBP classically associated to the germline and expressed in planarian neoblasts. Although little data exists, it

Please cite this article in press as: S. Krishna, et al., Post-transcriptional regulation in planarian stem cells, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.013

G Model

ARTICLE IN PRESS

YSCDB-2586; No. of Pages 10

S. Krishna et al. / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

4

Fig. 1. Overview of post-transcriptional regulation. Transcription of pre-mRNAs takes place in the nucleus. The maturation of an mRNA involves its capping, splicing, cleavage and polyadenylation. mRNAs are then exported out of the nucleus where they can be further localised, repressed, degraded or translated. All these processes are subject to regulation.

was shown that its knockdown induces neoblast loss and regeneration defects in Dugesia japonica [25] (Table 1). Many other RBPs are enriched in planarian neoblasts and are essential for neoblasts maintenance and regeneration [53]. In many of these cases though the mechanistic details on how these proteins functionally interact with others and with RNA to enable neoblasts to self-maintain and differentiate, and regeneration to proceed are still unknown. 6. Inside of the nucleus 6.1. The regulation of alternative splicing Perhaps the gene that we best understand at the mechanistic level is Smed-bruno-like, or bruli. Bruno genes typically encode two RRM domains and belong to the CELF family of RNA-binding proteins [54]. Bruno genes have been found in the germ line of Drosophila or mammals. In planarians, a Bruno homologue is also very highly expressed in planarians stem cells [27]. Its knockdown leads to neoblast loss and regeneration abrogation (Table 1). Members of the CELF family are known to be involved in the regulation of alternative splicing in different organisms [54]. Splicing is the process by which introns are removed from the pre-mRNA. The spliceosome, a large complex made of protein and RNA, performs this function [55]. Alternative splicing is the differential inclusion or exclusion of a given exon or set of exons in a transcript. This process is influenced by binding of several RBPs to the pre-mRNA, although it is still unclear how this RBPs influence the spliceosome. It results in different mRNA isoforms which can code for proteins that are different in structure, domain composition or

protein-protein interaction domains. Alternative splicing allows the encoding of different proteins by the same gene and therefore augments the complexity of the transcriptome. Planarian stem cells have a characteristic set of neoblast specific isoforms (Fig. 2) [38]. These isoforms and their neoblast specificity are conserved in different planarian species, indicating their functional relevance. The major regulator of this alternative splicing program in neoblasts is bruli: when bruli is knocked down by RNAi, neoblasts aberrantly express differentiated-cell specific isoforms. Bruli is needed in neoblasts both to enhance the inclusion of neoblast specific exons as well as to repress the inclusion of differentiated cell specific exons. Alternative exons enhanced by bruli often contain the BRULI binding motifs downstream of the regulated exon, showing that this regulation is likely exerted directly by the BRULI protein binding to these regions of the pre-mRNAs. Very interestingly, MBNL RBPs play an antagonistic role in planarians. Mbnl genes are expressed in differentiated cells and regulate an overlapping set of exons in an opposing way: when mbnl genes are knockdown planarian differentiated cells express neoblast specific mRNA isoforms (Table 1). Conversely, MBNL binding motifs are found enriched in differentiated cell specific exons that are enhanced by MBNL. The modes of action of BRULI and MBNL are thus like those of other RBPs in mammals. Bruli and mbnl inhibition have opposing effects as well at the phenotypic level. Strikingly, MBNL proteins are negative regulators of a set of exons that is differential of mammalian Embryonic Stem cells and induced Pluripotent Stem cells [56]. MBNL proteins thus have the same function in stem cell differentiation in both planarian and mammals. CELF proteins have been found to be antagonistic to

Please cite this article in press as: S. Krishna, et al., Post-transcriptional regulation in planarian stem cells, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.013

G Model YSCDB-2586; No. of Pages 10

ARTICLE IN PRESS S. Krishna et al. / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

5

Fig. 2. Alternative splicing in planarian stem cells. A set of alternative exons is characteristic of stem cell transcriptomes (red exons) and differentiated cell transcriptomes (blue exons). The RNA binding proteins BRULI and the different orthologs of MBNL regulate this set of exons. In neoblasts, BRULI enhances the inclusion of neoblast exons (in red) and represses the inclusion of differentiated cell exons in the mRNA. In contrast, in differentiated cells, MBNL orthologs repress the inclusion of neoblast exons and enhance the inclusion of differentiated cell exons.

MBNL proteins in mammalian models as well. Thus, CELF vs. MBNL antagonism is likely an ancient, universal mechanism of stem cell regulation. Several other arguments lend support to this notion. For instance, a Bruno gene was found to be expressed in poriferan stem cells -the so called archeocytes- while an MBNL gene was found enriched in differentiated cells instead [57]. These observations point to a scenario in which CELF and MBNL proteins antagonised to regulate the alternative splicing of stem cell and differentiated cell specific isoforms in the most basal metazoans and this regulation has been conserved for hundreds of millions of years in both planarians and mammals. A very enigmatic and understudied aspect of posttranscriptional regulation of planarian stem cells is the surprising prevalence of intron retention. This process consists in the presence of unspliced introns in the mature mRNA. Often these mRNAs are rendered invalid for protein production, due to in-frame stop codons present in the intron. Intron retention is an understudied process that has often been dismissed or regarded as expression noise. However, recent studies highlight its prevalence and regulated nature [58–61]. Programmed intron retention events can affect the transcript and its protein output in different ways. It is most commonly thought to induce nonsense mediated decay (NMD), a process by which incorrectly spliced transcripts are degraded. However, other responses are conceivable depending on the RBPs that bind to the retained introns. More than a hundred transcripts expressed in planarian neoblasts retain introns that are effectively spliced out in differentiated cells. A large portion of these retention events are regulated by bruli. Multiple introns are also retained in mammalian ESCs [58]. It is unknown if other invertebrate or vertebrate stem cells express mRNAs with similarly retained introns. The significance of this regulation remains unknown. Retained introns have conserved sequences [58,59].

Interestingly, the regulation of intron retention has recently been linked to PRMT5, a protein that methylates the arginine residues that are recognized by Tudor domains [62]. Future work will elucidate on the role of intron retention and its relationship with stem cells. 6.2. The regulation of alternative polyadenylation The 3 untranslated Regions (UTR) of the mRNA are “hotspots” for several post-transcriptional regulatory process. This region harbors binding sites for several RNA binding proteins, small RNAs and other post-transcriptional regulators. The lengths of the 3 UTRs therefore would be critical in dictating the fate of the mRNA. These lengths are determined by multi-protein complex that recognize a polyadenylation signals (PAS, canonically a hexameric sequence “AAUAAA”), cleave the UTR downstream of the PAS and adds polyA sequence thereafter. The polyadenylation complex involves sub-protein complexes; the CPSF (cleavage and polyadenylation specificity factor, that binds to the PAS), CstF (cleavage stimulation factor, that binds to U/GU rich region downstream of PAS), CFI and CFII (Cleavage factors I and II) and the PolyA Polymerase (PAP). It has been increasingly evident in the recent years that several mRNAs are alternately polyadenylated (ApA) [63]. Alternate polyadenylation refers to differential polyadenylation of a transcript as a consequence of the presence of two or more polyA signals within the transcript. It has been documented that over 50% of eukaryotic transcripts exhibit ApA. A genome-wide search for PAS in planaria identified “AAUAAA”, the canonical PAS to be the dominant PAS constituting 40% of all observed PAS. Over 44% of all the planarian transcripts exhibit at least a dual PAS thus strengthening the possibility of ApAmediated regulation [64]. ApA events, which determine the length

Please cite this article in press as: S. Krishna, et al., Post-transcriptional regulation in planarian stem cells, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.013

G Model YSCDB-2586; No. of Pages 10

ARTICLE IN PRESS S. Krishna et al. / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

6

of the 3 UTR was shown to be crucial for cell fate decisions. For example, it has been observed that highly proliferative cells often express transcripts with shorter 3 UTRs [65–67]. In proliferative cells an up-regulation of 3 processing factors CPSF and CstF was shown to be critical for the selection of proximal poly (A) signal over the distal resulting in the shorter 3 UTR [65]. In contrast, differentiating cells tend to have longer 3 UTRs as observed during embryonic development and myogenesis [68]. The identification of two or more PAS in thousands of neoblast-enriched transcripts suggest that a similar mechanism might exist in planaria that could be important for neoblast function and differentiation [64]. Inclusion or exclusion of sequences within the 3 UTR will determine the binding of several cis regulatory elements, which influences the gene expression. In planaria, it was observed that both the proximal and the distal 3 UTR exhibit hundreds of miRNA binding sites [64]. By selecting for the proximal PAS, the transcripts in planaria may exclude the binding of several miRNAs that potentially bind to the regions between the proximal and distal PAS. Existence of PAS within the coding region (CR) also changes the functionality of the protein either rendering it inactive or altering its activity, a process termed as CR-ApA. Around a hundred transcripts in planaria were shown to have PAS in the CDS suggesting a possibility of CR-ApA [64]. Further, the stoichiometry between the proteins involved in the polyadenylation and their interactions determine the selection of PAS leading to ApA. For example, overexpression of a CstF complex protein, CstF64, in B cells results in the switch in selection of PAS from proximal to distal thus producing a secretory form of IgM [69,70]. Although several of these complex proteins are expressed in planaria [64], a functional screen of these proteins may be necessary to understand their function in ApA and its biological significance.

7. Outside of the nucleus 7.1. Translational control and mRNA degradation Recent studies showed compelling evidence that eukaryotic translation may be tightly regulated by a cooperative interaction between RNA and protein complexes. Given the important role of translation regulators in controlling stem cell function and lineage determination, it becomes imperative to study the role of translation regulation in planarian regeneration. The process of translation can be broadly divided into three stages namely initiation, elongation and termination. Of these three stages, Initiation is the most rate-limiting and a hotspot of several regulatory mechanisms. Translation is initiated by the formation of eiF4F complex that involves the binding of eiF4E to the mRNA cap and to the scaffold protein eIF4G. A crucial step in the process of translation initiation is the interaction of the 3 and 5 ends of the mRNA, which involves the association of PABP (Poly A Binding Protein) with the eIF4G leading to circularization of the mRNA [71]. This interaction favours the recruitment of 40S ribosomal subunit along with the initiator tRNA (itRNA-Met) forming the 43S pre-initiation complex. Further studies also suggested an involvement of PABP in 60S recruitment and also translation termination [72,73]. Several other functions have also been attributed to PABP such as mRNA stability, RNA import and miRNA binding. The planarian genome has two homologs of PABP, namely Pabpc-1 and Pabpc-2 [53,74]. Functional characterization of Smed-Pabpc-1 and Smed-Pabpc-2 revealed distinct phenotypes suggesting different roles for these homologs in planaria. While the knockdown of Smed-Pabpc-1 resulted in a meiotic block of germline cells, the smed-Pabpc-2 knockdown showed more robust regeneration and homeostatic defect characterized by the lysis of the animals highlighting its important role in tissue homeostasis and regeneration [53,74,75] (Table 1).

Although most of the mRNAs contain a Poly A tail bound by PABPC, it has also been observed that the association of PABPC with specific regions of the transcripts could selectively influence the translational status of those transcripts. The association of PABPC with A rich elements on the 3 UTRs of certain transcripts, such as Ybx1 mRNA, is able to facilitate translation of these mRNAs [76]. Additional specificity may be gained by the interaction of PABP with RNA Binding proteins, such as DAZL, thereby activating translation of DAZL-bound transcripts [77,78]. Interestingly the knockdown of smed-pabpc2 in planaria revealed a translational down-regulation of a specific set of transcripts primarily involved in epidermal lineage specification, such as zfp-1. It may be interesting to probe the interactions of planarian PABPCs with different RNA-binding proteins to elucidate a mechanism for selective targeting by PABPC. This study also highlights also the critical role of translation regulation in controlling lineage specification during regeneration. The first step of mRNA degradation is deadenylation. The CCR4Not complex is the major deadenylating complex in eukaryotes. Upon differentiation, stem cells need to synthesize new mRNAs corresponding to their new differentiated functions whilst degrading stem cell specific mRNAs. The CCR4-Not complex is key to this activity. When Smed-not1, a gene encoding the largest subunit of the complex, is knockdown stem cells fail to differentiate and accumulate with larger amounts of stem cell mRNAs such as vasa, tudor and pcna [79] (Table 1). These genes also display longer poly-A tails compared to those of control animals. Animals fail to regenerate very early in this process, even at time points where neoblasts and their mitotic activity are abundantly detected. These observations point to a scenario in which the CCR4-Not is key to clear neoblasts mRNAs from the differentiated cells, and failure to do so prevents differentiation. In other organism it has been shown that some RBPs specifically bind to sequence motifs in the mRNAs and target these for degradation by tethering them to the CCR4-Not complex. Often these RBPs are also involved in translational repression. The RBPs that function to accomplish this function in planarians are still unresolved. Interestingly, the RNA-binding protein Smed-mex-3-1 [80] knockdown induces a very similar phenotype of accumulation of stem cell markers, abrogated differentiation, expansion of the stem cell pool and interrupted regeneration (Table 1). The direct targets and mechanisms by which mex-3 exerts this function are unknown. It is tantalizing to hypothesize that mex-3, a KH domain RBP, might tether its targets to the CCR4-Not complex. However, there are other possible scenarios. MEX-3 homologues have been shown to act as translational repressors in vertebrates and arthropods and it is a possibility that planarian MEX-3 induces translational repression in its targets. Other cytoplasmic RBPs might be involved in translational control or mRNA degradation. 8. Small RNAs in planarian stem cells With the advent of high-throughput sequencing, small RNA mediated post-transcriptional regulation has gained much prominence. Small RNAs, in sense, epitomize the RNA world hypothesis with their extensive influence and involvement at all, if not, most stages of the central dogma of life. Several species of small RNAs such as endo siRNAs, miRNAs, piRNAs, tRNA-derived small RNAs etc. have been identified and were shown to critically regulate various biological process. Of the several small RNA species, most efforts have been directed towards the understanding of microRNAs (miRNAs) and the piwi-interacting RNAs (piRNAs). High-throughput sequencing of small RNAs from planaria revealed that small RNAs in planaria belong predominantly to two main classes of small RNAs; miRNAs and piRNAs. miRNAs are ∼21 nt small non-coding RNAs that regulate gene expression by binding to 3 UTR of a transcript resulting in degra-

Please cite this article in press as: S. Krishna, et al., Post-transcriptional regulation in planarian stem cells, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.013

G Model YSCDB-2586; No. of Pages 10

ARTICLE IN PRESS S. Krishna et al. / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

7

Fig. 3. Small RNAs in planaria. miRNA biogenesis in planaria may be conserved as they express all the proteins required for miRNA production. miRNAs in association with RISC complex regulate target transcripts by either degradation or translational repression. The expression of miRNAs in different cell populations suggest important role for miRNAs in various biological process. miR-124 family of miRNAs facilitate neural patterning by repressing polarity genes such as slit, notch-2 and dscam. miRNAs also play crucial roles in planarian regeneration characterised by waves of miRNA expression correlating with the various cellular process during regeneration. Planarians also express the orthologs of the three piwi proteins involved in piRNA biogenesis; Smedwi-2 ortholog of Piwi; Smedwi-1/Semdwi-3 othrologs of Aubergine/Ago3. While all of them are predominatly expressd in the neoblasts, Smedwi-2 is restricted to nucleus and functions by repressesing transposable elements. Smedwi-1 and smedwi-3 are cytoplasmic proteins likely to function in the ping-pong piRNA biogenesis cycle. Planarian piwi proteins also associate with piRNAs that are derived from exons suggesting an alternate function. Smedwi-1 and smedwi-3 are also important for chromatoid body function and localisation of transcripts.

dation or translational silencing. The importance of miRNAs in regulation of cellular differentiation, cell fate decisions and regeneration in other organisms [81], make these small RNAs prime post-transcriptional regulators in planarian regeneration. miRNA biogenesis begins with the RNA Polymerase II dependent transcription of a polyadenylated transcript called the primary miRNA (Pri-miRNA). Drosha along with its partner DGCR8 cleaves the Pri-miRNA to generate the precursor miRNA (pre-miRNA). The premiRNA is exported to the cytoplasm by Exportin V – Ran GTPase system. In the cytoplasm, Dicer, an RNase III domain-containing enzyme, further processes the pre-miRNA to yield the 21–22 nt mature miRNA. Following the Dicer cleavage, the mature miRNA associates with Argonaute proteins, mainly Ago2, to form an effector complex termed as RNA induced silencing complex (RISC). The specificity in miRNA action is achieved by the base complementarity between 2–8 nt of the miRNA (seed region) and the 3 UTR of the target transcript. Systematic knockout studies by several groups have emphasized the importance for miRNA pathways in various biological contexts. Knockout of key enzymes involved in miRNA biogenesis such as Dicer, Drosha, DGCR8 and Ago2 led to embryonic lethality in mice. Many of these genes that are important for miRNA biogenesis have also been identified in planarians

[5,82]. The importance of miRNA mediated regulation in planaria was emphasized by the knockdown of Ago2 that led to regeneration defects and reduction in neoblasts resulting in the lysis of the animal [53,82] (Table 1). These observations suggest an important role for miRNA pathways in planarian stem cells and regeneration. Initial studies identified 126 miRNAs expressed in S. mediterranea of which 48 are unique to S. mediterranea [83,84]. A comprehensive cell-sorting based study by Sasidharan et al. identified only 6 miRNAs enriched in neoblasts compared to 10 miRNAs each in progenitors (X2) and differentiated cells (Xins) [85]. Although majority of the miRNAs are common between various cell types, it may be possible that a particular miRNA may regulate different targets in different cell types. Additionally, with recent reports suggesting cellular diversity within the stem cell pool [19], identification of miRNAs in different neoblast classes will offer a better understanding of miRNA mediated regulation in planarian stem cells. It is interesting to note that a well-characterized differentiation specific miRNA, let-7a, is enriched in the neoblast population suggesting alternative roles for this miRNA in planaria. Conversely, lin-28, a repressor of let-7, generally expressed in stem cells is enriched in differentiated cell population in planarians [84].

Please cite this article in press as: S. Krishna, et al., Post-transcriptional regulation in planarian stem cells, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.013

G Model YSCDB-2586; No. of Pages 10 8

ARTICLE IN PRESS S. Krishna et al. / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

miRNAs also play critical roles in planarian regeneration and development. Similar to the gene expression patterns observed by Wenemoser et al. [86], miRNA expression can also be correlated to the gene expression patterns – Category 1 miRNAs, that are expressed throughout regeneration possibly involved in homeostasis; Category 2 miRNAs, that are enriched in early time-points of regeneration (3 h–24 h) possibly critical for wound healing process and Category 3 miRNAs expressed during late regeneration (3–7 dpa) may be involved in development of organs (Fig. 3). Although the early regeneration processes, such as wound healing, are common to both anterior and posterior regeneration, the later stages of regeneration vary owing to the complex structures that exist in the anterior region. This is exemplified by the expression of Category 3 miRNAs observed predominantly in the anterior region. For example, expression profiles of a category 3 miRNA, miR124c correlate with the time-course of brain development during planarian regeneration. Interestingly, miR124 has been previously reported in several organisms to be a critical regulator for neurogenesis and neuronal differentiation. However, functional characterization of a Category 3 miRNA, miR124, revealed that in planaria, miR124 is essential for patterning of neural structures rather than neuronal differentiation itself [87] (Fig. 3). The recent annotation of 3 UTR [64] aided the identification of Slit-1, dscam and Notch-2 as targets for miR124. Further correlative studies between gene expression and miRNA expression changes during regeneration, followed by functional characterization may provide mechanistic insights in miRNA mediated regulation during regeneration. The other major class of small RNAs in planaria are the piRNAs. Piwi-interacting RNAs as the name suggest interact with a class of proteins called PIWI proteins, targeting invasive transposons thereby providing genomic stability. piRNAs are generally 26–32 nt long and are mainly expressed in germline cells [88]. Knockdown of piwi proteins in Drosophila and mouse causes severe defects in germline stem cells resulting in aberrant germline development and infertility. Initial studies identified 3 PIWI proteins in Drosophila to which piRNAs associate; Aubergine (Aub), Ago3 and Piwi. While Ago3 and Aubergine are expressed mainly in the germline stem cells, the expression of PIWI was reported in both germline stem cells and somatic follicular cells. Subsequent pulldown studies proposed two models for the biogenesis for piRNAs based on the sequence signatures of the piRNAs that these proteins associated with [89,90]. The ping-pong model of piRNA biogenesis suggests a cyclic amplification of the piRNA pool wherein the primary pIRNA transcript transcribed from the piRNA cluster is processed into a’ 32 nt primary piRNA which is recognized by Aub. The primary piRNA then base complements with the transposon transcript, following which the transcript is cleaved by Aub and an exonuclease to form the ∼32 nt secondary piRNA. The secondary piRNA associates with Ago3 and is recruited to process the primary piRNA transcript to aid in the production of primary piRNAs this forming a cycle. The second model involves the interaction of primary piRNAs arising from the piRNA cluster with Piwi to directly regulate the transposon transcript. Although these two models are widely accepted, a lot of key proteins involved in this complex processing remain unclear thus offering scope for research. Studies in S. mediterranea have identified 3 Piwi proteins; Smedwi-1, -2 and -3 primarily expressed in the neoblasts [5,91]. Knockdown studies have identified Smedwi-1 and Smedwi-2 to be critical to the functioning of stem cells (Table 1). These observations are supported by the recent knockdown of piwi proteins D. japonica wherein the knockdown of DjpiwiB and DjpiwiC led to the loss of neoblast and regeneration defects [92]. Deep sequencing analysis of small RNAs in S. mediterranea revealed only 20–30% of the small RNAs correspond to size ranges ∼32 nt, a typical size for piRNAs. These ∼31 nt small RNA molecules are regulated by smedwi-2 and -3 as supported by Northern hybridization analysis upon smedwi-2

and -3 knockdown [91]. Further, these ∼ 31 nt exhibited the signatures observed by piRNAs produced by the proposed ping-pong model for piRNA biogenesis with the sense strand showing a preference of ‘A’ at the 10th position and the anti-sense strand exhibiting a strong enrichment for ‘U’ at the 5 - end. The majority of piRNAs expressed in planaria map to distinct regions on the genome rather than to transposons thus resembling the pachytene piRNAs expressed in mammals. A recent report in D. japonica identified several ping-pong independent piRNAs mapping transposable elements associate with a nuclear Piwi protein DjPiwiB [92]. Given the signature of the piRNAs that DjpiwiB interacts with and the nuclear expression, it could be argued that DjpiwiB is similar to Piwi proteins of Drosophila while DjPiwiA and DjpiwiC represent the Ago3 and Aub proteins (Fig. 3). piRNAs in Drosophila and mammals are expressed in the germline but planarian piRNAs are mainly expressed in the neoblasts [83,91]. This expression is also observed in basal metazoans such as hydra, another classical model organism for regeneration [93]. The extreme capacity of these organisms to regenerate and to effectively reproduce asexually by regeneration with the help of stem cells could explain the presence of active piRNA pathway in these stem cell populations. Piwi genes and piRNAs are critical for the maintenance of genomic integrity. Since only a small percentage of piRNAs map to the transposable elements, it may be possible that apart from maintaining genomic integrity, piRNAs could aid in neoblast function. Additional functional characterization of specific piRNA clusters may be required to address this specific question. It is also worth noting that piRNAs in planaria, although a minor proportion, map to exonic regions [83,91]. Similar observations have also been made in hydra suggesting that in these basal metazoans, piRNA pathways may have evolved to regulate gene expression [93]. The localization of Histone H4 mRNA to chromatoid bodies, which is regulated by Smedwi-2 and Smedwi-3 also supports this hypothesis that piwi proteins and piRNA pathway may have evolved to regulate gene expression in basal metazoans [94] (Fig. 3). 9. Future perspectives The field of planarian stem cell biology is exploding thanks to recent advancements in transcriptomics and single cell technologies. Post-transcriptional gene regulation is a critical aspect of planarian, germ and mammalian stem cell biology. The large number of stem cells present in planarians and the ability to make functional studies in vivo make them an ideal model to study these processes. The implementation of biochemical techniques to elucidate RBP:target interactions would greatly help us in understanding the syntax of RNA regulation in stem cells. The combination of biochemical, functional and single cell studies is a very promising path to understand how stem cells differentiate into a myriad of cell types but self-maintain throughout the lifetime of the animal. Acknowledgments Part of the work in the review was supported by Wellcome/DBT India Alliance Intermediate fellowship awarded to DP. DP is supported by Swarna Jayanthi, DST, Fellowship. SK is supported by the funding from DBT Grant (BT/PR8655/AGR/36/759/2013). References [1] A.A. Aboobaker, Planarian stem cells: a simple paradigm for regeneration, Trends Cell Biol. 21 (5) (2011) 304–311. [2] J. Baguna, The planarian neoblast: the rambling history of its origin and some current black boxes, Int. J. Dev. Biol. 56 (1–3) (2012) 19–37.

Please cite this article in press as: S. Krishna, et al., Post-transcriptional regulation in planarian stem cells, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.013

G Model YSCDB-2586; No. of Pages 10

ARTICLE IN PRESS S. Krishna et al. / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

[3] J.C. Rink, Stem cell systems and regeneration in planaria, Dev. Genes Evol. 223 (1–2) (2013) 67–84. [4] D.E. Wagner, I.E. Wang, P.W. Reddien, Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration, Science 332 (6031) (2011) 811–816. [5] P.W. Reddien, N.J. Oviedo, J.R. Jennings, J.C. Jenkin, A. Sanchez Alvarado, SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells, Science 310 (5752) (2005) 1327–1330. [6] T. Hayashi, M. Asami, S. Higuchi, N. Shibata, K. Agata, Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting, Dev. Growth Differ. 48 (6) (2006) 371–380. [7] P.A. Newmark, P.W. Reddien, F. Cebria, A. Sanchez Alvarado, Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians, Proc. Natl. Acad. Sci. U. S. A. 100 (Suppl. (1)) (2003) 11861–11865. [8] S.M. Robb, K. Gotting, E. Ross, A. Sanchez Alvarado, SmedGD 2.0: the Schmidtea mediterranea genome database, Genesis 53 (8) (2015) 535–546. [9] M.A. Grohme, S. Schloissnig, A. Rozanski, M. Pippel, G.R. Young, S. Winkler, H. Brandl, I. Henry, A. Dahl, S. Powell, M. Hiller, E. Myers, J.C. Rink, The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms, Nature 554 (7690) (2018) 56–61. [10] C. Adamidi, Y. Wang, D. Gruen, G. Mastrobuoni, X. You, D. Tolle, M. Dodt, S.D. Mackowiak, A. Gogol-Doering, P. Oenal, A. Rybak, E. Ross, A. Sanchez Alvarado, S. Kempa, C. Dieterich, N. Rajewsky, W. Chen, De novo assembly and validation of planaria transcriptome by massive parallel sequencing and shotgun proteomics, Genome Res. 21 (7) (2011) 1193–1200. [11] M.J. Blythe, D. Kao, S. Malla, J. Rowsell, R. Wilson, D. Evans, J. Jowett, A. Hall, V. Lemay, S. Lam, A.A. Aboobaker, A dual platform approach to transcript discovery for the planarian Schmidtea mediterranea to establish RNAseq for stem cell and regeneration biology, PLoS One 5 (12) (2010), e15617. [12] H. Brandl, H. Moon, M. Vila-Farre, S.Y. Liu, I. Henry, J.C. Rink, PlanMine - a mineable resource of planarian biology and biodiversity, Nucleic Acids Res. 44 (D1) (2016) D764–D773. [13] R.M. Labbe, M. Irimia, K.W. Currie, A. Lin, S.J. Zhu, D.D. Brown, E.J. Ross, V. Voisin, G.D. Bader, B.J. Blencowe, B.J. Pearson, A comparative transcriptomic analysis reveals conserved features of stem cell pluripotency in planarians and mammals, Stem Cells 30 (8) (2012) 1734–1745. [14] P. Onal, D. Grun, C. Adamidi, A. Rybak, J. Solana, G. Mastrobuoni, Y. Wang, H.P. Rahn, W. Chen, S. Kempa, U. Ziebold, N. Rajewsky, Gene expression of pluripotency determinants is conserved between mammalian and planarian stem cells, EMBO J. 31 (12) (2012) 2755–2769. [15] A.M. Resch, D. Palakodeti, Y.C. Lu, M. Horowitz, B.R. Graveley, Transcriptome analysis reveals strain-specific and conserved stemness genes in Schmidtea mediterranea, PLoS One 7 (4) (2012), e34447. [16] G. Rodriguez-Esteban, A. Gonzalez-Sastre, J.I. Rojo-Laguna, E. Salo, J.F. Abril, Digital gene expression approach over multiple RNA-seq data sets to detect neoblast transcriptional changes in Schmidtea mediterranea, BMC Genome 16 (2015) 361. [17] J. Solana, D. Kao, Y. Mihaylova, F. Jaber-Hijazi, S. Malla, R. Wilson, A. Aboobaker, Defining the molecular profile of planarian pluripotent stem cells using a combinatorial RNAseq, RNA interference and irradiation approach, Genome Biol. 13 (3) (2012), R19. [18] A.M. Molinaro, B.J. Pearson, In silico lineage tracing through single cell transcriptomics identifies a neural stem cell population in planarians, Genome Biol. 17 (2016) 87. [19] J.C. van Wolfswinkel, D.E. Wagner, P.W. Reddien, Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment, Cell Stem Cell 15 (3) (2014) 326–339. [20] O. Wurtzel, L.E. Cote, A. Poirier, R. Satija, A. Regev, P.W. Reddien, A generic and Cell-type-specific wound response precedes regeneration in planarians, Dev. Cell 35 (5) (2015) 632–645. [21] O. Wurtzel, I.M. Oderberg, P.W. Reddien, Planarian epidermal stem cells respond to positional cues to promote cell-type diversity, Dev. Cell 40 (5) (2017) 491–504, e5. [22] C.T. Fincher, O. Wurtzel, T. de Hoog, K.M. Kravarik, P.W. Reddien, Cell type transcriptome atlas for the planarian Schmidtea mediterranea, Science 360 (6391) (2018). [23] M. Plass, J. Solana, F.A. Wolf, S. Ayoub, A. Misios, P. Glazar, B. Obermayer, F.J. Theis, C. Kocks, N. Rajewsky, Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics, Science 360 (6391) (2018). [24] N. Shibata, Y. Umesono, H. Orii, T. Sakurai, K. Watanabe, K. Agata, Expression of vasa(vas)-related genes in germline cells and totipotent somatic stem cells of planarians, Dev. Biol. 206 (1) (1999) 73–87. [25] A. Salvetti, L. Rossi, A. Lena, R. Batistoni, P. Deri, G. Rainaldi, M.T. Locci, M. Evangelista, V. Gremigni, DjPum, a homologue of Drosophila Pumilio, is essential to planarian stem cell maintenance, Development 132 (8) (2005) 1863–1874. [26] J. Solana, P. Lasko, R. Romero, Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal, Dev. Biol. 328 (2) (2009) 410–421. [27] T. Guo, A.H. Peters, P.A. Newmark, A bruno-like gene is required for stem cell maintenance in planarians, Dev. Cell 11 (2) (2006) 159–169. [28] J. Solana, Closing the circle of germline and stem cells: the primordial stem cell hypothesis, Evodevo 4 (1) (2013) 2. [29] S.J. Coward, Chromatoid bodies in somatic cells of the planarian: observations on their behavior during mitosis, Anat. Rec. 180 (3) (1974) 533–545.

9

[30] R. Lehmann, Germ plasm biogenesis–an oskar-centric perspective, Curr. Top. Dev. Biol. 116 (2016) 679–707. [31] S. Strome, D. Updike, Specifying and protecting germ cell fate, Nat. Rev. Mol. Cell Biol. 16 (7) (2015) 406–416. [32] E. Fernandez-Taboada, S. Moritz, D. Zeuschner, M. Stehling, H.R. Scholer, E. Salo, L. Gentile, Smed-SmB, a member of the LSm protein superfamily, is essential for chromatoid body organization and planarian stem cell proliferation, Development 137 (7) (2010) 1055–1065. [33] M. Yoshida-Kashikawa, N. Shibata, K. Takechi, K. Agata, DjCBC-1, a conserved DEAD box RNA helicase of the RCK/p54/Me31B family, is a component of RNA-protein complexes in planarian stem cells and neurons, Dev. Dyn. 236 (12) (2007) 3436–3450. [34] N. Rebscher, F. Zelada-Gonzalez, T.U. Banisch, F. Raible, D. Arendt, Vasa unveils a common origin of germ cells and of somatic stem cells from the posterior growth zone in the polychaete platynereis dumerilii, Dev. Biol. 306 (2) (2007) 599–611. [35] C.E. Juliano, E. Voronina, C. Stack, M. Aldrich, A.R. Cameron, G.M. Wessel, Germ line determinants are not localized early in sea urchin development, but do accumulate in the small micromere lineage, Dev. Biol. 300 (1) (2006) 406–415. [36] C.E. Juliano, S.Z. Swartz, G.M. Wessel, A conserved germline multipotency program, Development 137 (24) (2010) 4113–4126. [37] S. Gerstberger, M. Hafner, T. Tuschl, A census of human RNA-binding proteins, Nat. Rev. Genet. 15 (12) (2014) 829–845. [38] J. Solana, M. Irimia, S. Ayoub, M.R. Orejuela, V. Zywitza, M. Jens, J. Tapial, D. Ray, Q. Morris, T.R. Hughes, B.J. Blencowe, N. Rajewsky, Conserved functional antagonism of CELF and MBNL proteins controls stem cell-specific alternative splicing in planarians, eLife 5 (2016), e16797, pii:. [39] R.E. Halbeisen, A. Galgano, T. Scherrer, A.P. Gerber, Post-transcriptional gene regulation: from genome-wide studies to principles, Cell. Mol. Life Sci. 65 (5) (2008) 798–813. [40] G.R. Gronland, A. Ramos, The devil is in the domain: understanding protein recognition of multiple RNA targets, Biochem. Soc. Trans. 45 (6) (2017) 1305–1311. [41] M. Hafner, M. Landthaler, L. Burger, M. Khorshid, J. Hausser, P. Berninger, A. Rothballer, M. Ascano, A.C. Jungkamp, M. Munschauer, A. Ulrich, G.S. Wardle, S. Dewell, M. Zavolan, T. Tuschl, PAR-CliP–a method to identify transcriptome-wide the binding sites of RNA binding proteins, J. Vis. Exp. (41) (2010). [42] J. Konig, K. Zarnack, G. Rot, T. Curk, M. Kayikci, B. Zupan, D.J. Turner, N.M. Luscombe, J. Ule, iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution, Nat. Struct. Mol. Biol. 17 (7) (2010) 909–915. [43] J. Ule, K.B. Jensen, M. Ruggiu, A. Mele, A. Ule, R.B. Darnell, CLIP identifies Nova-regulated RNA networks in the brain, Science 302 (5648) (2003) 1212–1215. [44] D. Ray, H. Kazan, K.B. Cook, M.T. Weirauch, H.S. Najafabadi, X. Li, S. Gueroussov, M. Albu, H. Zheng, A. Yang, H. Na, M. Irimia, L.H. Matzat, R.K. Dale, S.A. Smith, C.A. Yarosh, S.M. Kelly, B. Nabet, D. Mecenas, W.M. Li, R.S. Laishram, M. Qiao, H.D. Lipshitz, F. Piano, A.H. Corbett, R.P. Carstens, B.J. Frey, R.A. Anderson, K.W. Lynch, L.O.F. Penalva, E.P. Lei, A.G. Fraser, B.J. Blencowe, Q.D. Morris, T.R. Hughes, A compendium of RNA-binding motifs for decoding gene regulation, Nature 499 (7457) (2013) 172–177. [45] B. Raj, B.J. Blencowe, Alternative splicing in the mammalian nervous system: recent insights into mechanisms and functional roles, Neuron 87 (1) (2015) 14–27. [46] P. Lasko, The DEAD-box helicase vasa: evidence for a multiplicity of functions in RNA processes and developmental biology, Biochim. Biophys. Acta 1829 (8) (2013) 810–816. [47] C.F. Bourgeois, F. Mortreux, D. Auboeuf, The multiple functions of RNA helicases as drivers and regulators of gene expression, Nat. Rev. Mol. Cell Biol. 17 (7) (2016) 426–438. [48] J. Solana, R. Romero, SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development, Int. J. Biol. Sci. 5 (1) (2009) 64–73. [49] D.E. Wagner, J.J. Ho, P.W. Reddien, Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis, Cell Stem Cell 10 (3) (2012) 299–311. [50] S. Gayatri, M.T. Bedford, Readers of histone methylarginine marks, Biochim. Biophys. Acta 1839 (8) (2014) 702–710. [51] M. Handberg-Thorsager, E. Salo, The planarian nanos-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration, Dev. Genes Evol. 217 (5) (2007) 403–411. [52] Y. Wang, R.M. Zayas, T. Guo, P.A. Newmark, Nanos function is essential for development and regeneration of planarian germ cells, Proc. Natl. Acad. Sci. U. S. A. 104 (14) (2007) 5901–5906. [53] L. Rouhana, N. Shibata, O. Nishimura, K. Agata, Different requirements for conserved post-transcriptional regulators in planarian regeneration and stem cell maintenance, Dev. Biol. 341 (2) (2010) 429–443. [54] T. Dasgupta, A.N. Ladd, The importance of CELF control: molecular and biological roles of the CUG-BP, elav-like family of RNA-binding proteins, Wiley Interdiscip. Rev. RNA 3 (1) (2012) 104–121. [55] Y. Shi, The spliceosome: a protein-directed metalloribozyme, J. Mol. Biol. 429 (17) (2017) 2640–2653. [56] H. Han, M. Irimia, P.J. Ross, H.K. Sung, B. Alipanahi, L. David, A. Golipour, M. Gabut, I.P. Michael, E.N. Nachman, E. Wang, D. Trcka, T. Thompson, D. O’Hanlon, V. Slobodeniuc, N.L. Barbosa-Morais, C.B. Burge, J. Moffat, B.J. Frey, A. Nagy, J. Ellis, J.L. Wrana, B.J. Blencowe, MBNL proteins repress

Please cite this article in press as: S. Krishna, et al., Post-transcriptional regulation in planarian stem cells, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.013

G Model YSCDB-2586; No. of Pages 10 10

[57]

[58] [59]

[60] [61]

[62]

[63] [64]

[65]

[66]

[67]

[68]

[69]

[70]

[71] [72]

[73]

[74]

[75]

ARTICLE IN PRESS S. Krishna et al. / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

ES-cell-specific alternative splicing and reprogramming, Nature 498 (7453) (2013), 241-+. A. Alie, T. Hayashi, I. Sugimura, M. Manuel, W. Sugano, A. Mano, N. Satoh, K. Agata, N. Funayama, The ancestral gene repertoire of animal stem cells, Proc. Natl. Acad. Sci. U. S. A. 112 (51) (2015), E7093–100. P.L. Boutz, A. Bhutkar, P.A. Sharp, Detained introns are a novel, widespread class of post-transcriptionally spliced introns, Genes Dev. 29 (1) (2015) 63–80. U. Braunschweig, N.L. Barbosa-Morais, Q. Pan, E.N. Nachman, B. Alipanahi, T. Gonatopoulos-Pournatzis, B. Frey, M. Irimia, B.J. Blencowe, Widespread intron retention in mammals functionally tunes transcriptomes, Genome Res. 24 (11) (2014) 1774–1786. A.G. Jacob, C.W.J. Smith, Intron retention as a component of regulated gene expression programs, Hum. Genet. 136 (9) (2017) 1043–1057. D.P. Vanichkina, U. Schmitz, J.J. Wong, J.E.J. Rasko, Challenges in defining the role of intron retention in normal biology and disease, Semin. Cell Dev. Biol. 75 (2018) 40–49. C.J. Braun, M. Stanciu, P.L. Boutz, J.C. Patterson, D. Calligaris, F. Higuchi, R. Neupane, S. Fenoglio, D.P. Cahill, H. Wakimoto, N.Y.R. Agar, M.B. Yaffe, P.A. Sharp, M.T. Hemann, J.A. Lees, Coordinated splicing of regulatory detained introns within oncogenic transcripts creates an exploitable vulnerability in malignant glioma, Cancer Cell 32 (4) (2017) 411–426, e11. B. Tian, J.L. Manley, Alternative polyadenylation of mRNA precursors, Nat. Rev. Mol. Cell Biol. 18 (1) (2017) 18–30. V. Lakshmanan, D. Bansal, J. Kulkarni, D. Poduval, S. Krishna, V. Sasidharan, P. Anand, A. Seshasayee, D. Palakodeti, Genome-wide analysis of polyadenylation events in Schmidtea mediterranea, G3 (Bethesda) 6 (10) (2016) 3035–3048. Z. Ji, B. Tian, Reprogramming of 3’ untranslated regions of mRNAs by alternative polyadenylation in generation of pluripotent stem cells from different cell types, PLoS One 4 (12) (2009), e8419. C. Mayr, D.P. Bartel, Widespread shortening of 3’UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells, Cell 138 (4) (2009) 673–684. R. Sandberg, J.R. Neilson, A. Sarma, P.A. Sharp, C.B. Burge, Proliferating cells express mRNAs with shortened 3’ untranslated regions and fewer microRNA target sites, Science 320 (5883) (2008) 1643–1647. Z. Ji, J.Y. Lee, Z. Pan, B. Jiang, B. Tian, Progressive lengthening of 3’ untranslated regions of mRNAs by alternative polyadenylation during mouse embryonic development, Proc. Natl. Acad. Sci. U. S. A. 106 (17) (2009) 7028–7033. Y. Takagaki, J.L. Manley, Levels of polyadenylation factor CstF-64 control IgM heavy chain mRNA accumulation and other events associated with B cell differentiation, Mol. Cell 2 (6) (1998) 761–771. Y. Takagaki, R.L. Seipelt, M.L. Peterson, J.L. Manley, The polyadenylation factor CstF-64 regulates alternative processing of IgM heavy chain pre-mRNA during B cell differentiation, Cell 87 (5) (1996) 941–952. D.J. Goss, F.E. Kleiman, Poly(A) binding proteins: are they all created equal? Wiley Interdiscip. Rev. RNA 4 (2) (2013) 167–179. N. Amrani, S. Ghosh, D.A. Mangus, A. Jacobson, Translation factors promote the formation of two states of the closed-loop mRNP, Nature 453 (7199) (2008) 1276–1280. A. Kahvejian, Y.V. Svitkin, R. Sukarieh, M.N. M’Boutchou, N. Sonenberg, Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms, Genes Dev. 19 (1) (2005) 104–113. D. Bansal, J. Kulkarni, K. Nadahalli, V. Lakshmanan, S. Krishna, V. Sasidharan, J. Geo, S. Dilipkumar, R. Pasricha, A. Gulyani, S. Raghavan, D. Palakodeti, Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea, Development 144 (17) (2017) 3066–3079. Y. Wang, J.M. Stary, J.E. Wilhelm, P.A. Newmark, A functional genomic screen in planarians identifies novel regulators of germ cell development, Genes Dev. 24 (18) (2010) 2081–2092.

[76] D.N. Lyabin, I.A. Eliseeva, O.V. Skabkina, L.P. Ovchinnikov, Interplay between y-box-binding protein 1 (YB-1) and poly(A) binding protein (PABP) in specific regulation of YB-1 mRNA translation, RNA Biol. 8 (5) (2011) 883–892. [77] B. Collier, B. Gorgoni, C. Loveridge, H.J. Cooke, N.K. Gray, The DAZL family proteins are PABP-binding proteins that regulate translation in germ cells, EMBO J. 24 (14) (2005) 2656–2666. [78] N. Reynolds, B. Collier, K. Maratou, V. Bingham, R.M. Speed, M. Taggart, C.A. Semple, N.K. Gray, H.J. Cooke, Dazl binds in vivo to specific transcripts and can regulate the pre-meiotic translation of Mvh in germ cells, Hum. Mol. Genet. 14 (24) (2005) 3899–3909. [79] J. Solana, C. Gamberi, Y. Mihaylova, S. Grosswendt, C. Chen, P. Lasko, N. Rajewsky, A.A. Aboobaker, The CCR4-NOT complex mediates deadenylation and degradation of stem cell mRNAs and promotes planarian stem cell differentiation, PLoS Genet. 9 (12) (2013), e1004003. [80] S.J. Zhu, S.E. Hallows, K.W. Currie, C. Xu, B.J. Pearson, A mex3 homolog is required for differentiation during planarian stem cell lineage development, eLife 4 (2015). [81] M.S. Ebert, P.A. Sharp, Roles for microRNAs in conferring robustness to biological processes, Cell 149 (3) (2012) 515–524. [82] Y.Q. Li, A. Zeng, X.S. Han, C. Wang, G. Li, Z.C. Zhang, J.Y. Wang, Y.W. Qin, Q. Jing, Argonaute-2 regulates the proliferation of adult stem cells in planarian, Cell Res. 21 (12) (2011) 1750–1754. [83] M.R. Friedlander, C. Adamidi, T. Han, S. Lebedeva, T.A. Isenbarger, M. Hirst, M. Marra, C. Nusbaum, W.L. Lee, J.C. Jenkin, A. Sanchez Alvarado, J.K. Kim, N. Rajewsky, High-resolution profiling and discovery of planarian small RNAs, Proc. Natl. Acad. Sci. U. S. A. 106 (28) (2009) 11546–11551. [84] Y.C. Lu, M. Smielewska, D. Palakodeti, M.T. Lovci, S. Aigner, G.W. Yeo, B.R. Graveley, Deep sequencing identifies new and regulated microRNAs in Schmidtea mediterranea, RNA 15 (8) (2009) 1483–1491. [85] V. Sasidharan, Y.C. Lu, D. Bansal, P. Dasari, D. Poduval, A. Seshasayee, A.M. Resch, B.R. Graveley, D. Palakodeti, Identification of neoblast- and regeneration-specific miRNAs in the planarian Schmidtea mediterranea, RNA 19 (10) (2013) 1394–1404. [86] D. Wenemoser, S.W. Lapan, A.W. Wilkinson, G.W. Bell, P.W. Reddien, A molecular wound response program associated with regeneration initiation in planarians, Genes Dev. 26 (9) (2012) 988–1002. [87] V. Sasidharan, S. Marepally, S.A. Elliott, S. Baid, V. Lakshmanan, N. Nayyar, D. Bansal, A. Sanchez Alvarado, P.K. Vemula, D. Palakodeti, The miR-124 family of microRNAs is crucial for regeneration of the brain and visual system in the planarian Schmidtea mediterranea, Development 144 (18) (2017) 3211–3223. [88] Y.W. Iwasaki, M.C. Siomi, H. Siomi, PIWI-interacting RNA: its biogenesis and functions, Annu. Rev. Biochem. 84 (2015) 405–433. [89] J. Brennecke, A.A. Aravin, A. Stark, M. Dus, M. Kellis, R. Sachidanandam, G.J. Hannon, Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila, Cell 128 (6) (2007) 1089–1103. [90] L.S. Gunawardane, K. Saito, K.M. Nishida, K. Miyoshi, Y. Kawamura, T. Nagami, H. Siomi, M.C. Siomi, A slicer-mediated mechanism for repeat-associated siRNA 5’ end formation in Drosophila, Science 315 (5818) (2007) 1587–1590. [91] D. Palakodeti, M. Smielewska, Y.C. Lu, G.W. Yeo, B.R. Graveley, The PIWI proteins SMEDWI-2 and SMEDWI-3 are required for stem cell function and piRNA expression in planarians, RNA 14 (6) (2008) 1174–1186. [92] N. Shibata, M. Kashima, T. Ishiko, O. Nishimura, L. Rouhana, K. Misaki, S. Yonemura, K. Saito, H. Siomi, M.C. Siomi, K. Agata, Inheritance of a nuclear PIWI from pluripotent stem cells by somatic descendants ensures differentiation by silencing transposons in planarian, Dev. Cell 37 (3) (2016) 226–237. [93] S. Krishna, A. Nair, S. Cheedipudi, D. Poduval, J. Dhawan, D. Palakodeti, Y. Ghanekar, Deep sequencing reveals unique small RNA repertoire that is regulated during head regeneration in Hydra magnipapillata, Nucleic Acids Res. 41 (1) (2013) 599–616. [94] L. Rouhana, J.A. Weiss, R.S. King, P.A. Newmark, PIWI homologs mediate histone H4 mRNA localization to planarian chromatoid bodies, Development 141 (13) (2014) 2592–2601.

Please cite this article in press as: S. Krishna, et al., Post-transcriptional regulation in planarian stem cells, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.013