Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion during eukaryotic chromosomal replication, but also has poorly understood non-canonical functions. Here, in the model social amoeba Dictyostelium discoideum , we show that the protein encoded by tert has telomerase-like motifs, and regulates, non-canonically, important developmental processes. Expression levels of wild-type (WT) tert were biphasic, peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, forming small mounds. The mound-size defect was not induced when a KO mutant of countin (a master size-regulating gene) was treated with TERT inhibitors, but anti-countin antibodies did rescue size in the tert KO. Although, conditioned medium (CM) from countin mutants failed to rescue size in the tert KO, tert KO CM rescued the countin KO phenotype. These and additional observations indicate that TERT acts upstream of smlA/countin : (i) the observed expression levels of smlA and countin , being respectively lower and higher (than WT) in the tert KO; (ii) the levels of known size-regulation intermediates, glucose (low) and adenosine (high), in the tert mutant, and the size defect’s rescue by supplemented glucose or the adenosine-antagonist, caffeine; (iii) the induction of the size defect in the WT by tert KO CM and TERT inhibitors. The tert KO’s other defects (delayed aggregation, irregular streaming) were associated with changes to cAMP-regulated processes (e.g. chemotaxis, cAMP pulsing) and their regulatory factors (e.g. cAMP; acaA , carA expression). Overexpression of WT tert in the tert KO rescued these defects (and size), and restored a single cAMP signaling centre. Our results indicate that TERT acts in novel, non-canonical and upstream ways, regulating key developmental events in Dictyostelium .

11Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA 12 erosion during eukaryotic chromosomal replication, but also has poorly understood non-13 canonical functions. Here, in the model social amoeba Dictyostelium discoideum, we show 14 that the protein encoded by tert has telomerase-like motifs and regulates, non-canonically, 15 important developmental processes. Expression levels of wild-type (WT) tert were biphasic, 16 peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the 17 initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, 18 aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, leading 19 to small mounds. The mound-size defect was not induced when a KO mutant of countin (a 20 master size-regulating gene) was treated with TERT inhibitors but anti-countin antibodies did 21 rescue size in the tert KO. Further, conditioned medium from countin mutants failed to 22 rescue size in the tert KO, but the converse experiment worked. These and additional 23 the migration of cells that form each fruiting body is delayed and irregular. These results are 48 significant because they show, for the first time, that a telomerase can influence cell 49 migration and tissue size regulation, two processes involved in a wide range of cancers. 50 51 Introduction 52 Each time a chromosome replicates, it loses some DNA from each of its ends. This is not 53 necessarily problematic, because the chromosome is initially capped at each end by a 54 sacrificial strand of non-coding DNA, a telomere [1][2][3]. Further instances of replication, 55 however, can expose the coding DNA, unless the cell can keep repairing the shortened 56 telomeres, by the action of the enzyme complex, telomerase. Accordingly, telomerase, whose 57 main subunits comprise a reverse transcriptase (TERT), and the telomerase RNA component 58 (TERC) [4], has much significance in the biology and pathology of multicellular organisms. 59As somatic tissues age, for example, telomerase is downregulated, and the resulting telomeric 60 dysfunction can lead to chromosomal instability and various pathologies, including disrupted 61 pregnancies and cancer [5][6][7]. In other cases, the upregulation of telomerase is also associated 62 with, and a biomarker of, some cancers, because it allows the unchecked proliferation of 63 immortalised tumour cells [6, 8]. Telomerase also has many non-canonical roles, in which 64 telomere maintenance, or even telomerase activity, is not required [9, 10]. For example, 65 telomerase is known to have non-canonical roles in neuronal differentiation [11], RNA 66 silencing [12], enhanced mitochondrial function [13] and various cancers [9, 14]. 67Our understanding of telomeres and telomerase began, and has continued to develop, through 68 the study of model organisms such as Drosophila, Zea mays, Tetrahymena, yeast and mice 69 [2, 3,[15][16][17][18][19]]. One model system in which the possible roles of telomerase hav...