Caenorhabditis elegans period homolog lin-42 regulates the timing of heterochronic miRNA expression

Abstract

MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally via the 3′ UTR of target mRNAs and were first identified in the Caenorhabditis elegans heterochronic pathway. miRNAs have since been found in many organisms and have broad functions, including control of differentiation and pluripotency in humans. lin-4 and let-7-family miRNAs regulate developmental timing in C. elegans, and their proper temporal expression ensures cell lineage patterns are correctly timed and sequentially executed. Although much is known about miRNA biogenesis, less is understood about how miRNA expression is timed and regulated. lin-42, the worm homolog of the circadian rhythm gene period of flies and mammals, is another core component of the heterochronic gene pathway. lin-42 mutants have a precocious phenotype, in which later-stage programs are executed too early, but the placement of lin-42 in the timing pathway is unclear. Here, we demonstrate that lin-42 negatively regulates heterochronic miRNA transcription. let-7 and the related miRNA miR-48 accumulate precociously in lin-42 mutants. This defect reflects transcriptional misregulation because enhanced expression of both primary miRNA transcripts (pri-miRNAs) and a let-7 promoter::gfp fusion are observed. The pri-miRNA levels oscillate during larval development, in a pattern reminiscent of lin-42 expression. Importantly, we show that lin-42 is not required for this cycling; instead, peak amplitude is increased. Genetic analyses further confirm that lin-42 acts through let-7 family miRNAs. Taken together, these data show that a key function of lin-42 in developmental timing is to dampen pri-miRNAs levels, preventing their premature expression as mature miRNAs.M etazoan development uses both positional and temporal information to pattern life stages from the single-cell embryo to the reproductive adult. In Caenorhabditis elegans, the temporal component of postembryonic patterning is conveyed through the heterochronic gene regulatory circuit (see ref. 1 for review). A core theme of the heterochronic circuit is that a series of microRNA (miRNA) switches promote transitions from one stage to the next by negatively regulating key factors that direct stage-specific programs, thereby allowing successive temporal fates to be executed.Five conserved miRNAs, lin-4 and the let-7 family members, let-7, miR-48, miR-84, and miR-241, play prominent roles in the circuit (2-6). lin-4 appears first, midway through the L1 larval stage and guides the transition from the L1 to L2 stage (7). miR-48, miR-84, and miR-241 amass in the L2 and control the transition to the L3 stage, when let-7 levels rise dramatically and govern the switch to the L4. Mutations in these miRNAs cause the relevant transition to fail to advance. This process results in, for example, repetition of the L1-stage pattern in lin-4 mutants or L2-stage patterns in mir-48/84/241 mutants, consistent with the sequential expression of these miRNAs. Deciphering this temporal control mechanism then simplifies, to a...

Analysis of alin-42/periodNull Allele Implicates All Three Isoforms in Regulation ofCaenorhabditis elegansMolting and Developmental Timing

“…Prior results that link lin-42 activity to control of the proliferative division prompted assessment of seam cell number in lin-42 ( 0 ) animals. Notably, lin-42 negatively regulates expression of mir-48 (McCulloch and Rougvie 2014; Perales et al 2014; Van Wynsberghe et al 2014), a miRNA that when overexpressed, results in reduced seam cell number (Li et al 2005). In addition, when grown at the permissive temperature of 15°, lin-14 ( n179ts ); lin-42 ( n1089 ) animals skip the L2 stage proliferative division, a phenotype not observed in either single mutant (Liu 1990).…”

“…RNA was prepared from synchronized populations of wild-type animals collected at 2-hr intervals from 6 to 36 hr and transcript levels were assayed, using endogenous mlt-10 expression as an internal control to mark developmental time and monitor synchrony. mlt-10 message levels also oscillate, peaking ∼4 hr before each lethargus (Figure 4) (Frand et al 2005; McCulloch and Rougvie 2014). The expression patterns of the lin-42 transcripts were found to be highly similar to each other (Figure 4), peaking at approximately the same time and returning to a low basal level before each molt (∼4 hr after the mlt-10 peak), despite being derived from two different promoters.…”

“…lin-42 has a well-defined role as a negative transcriptional regulator of miRNA genes, including some that function in the heterochronic gene pathway (McCulloch and Rougvie 2014; Perales et al 2014; Van Wynsberghe et al 2014), potentially allowing lin-42 to indirectly coordinate translation of many downstream messenger RNAs. Initial chromatin immunoprecipitation coupled to sequencing (ChIP-seq) experiments have indeed found LIN-42 associated with chromatin at miRNA promoters, but in addition, LIN-42 is found near the transcription starts of protein-coding genes (Perales et al 2014).…”

“…For example, mutations in the heterochronic gene lin-42 cause a precocious phenotype, as demonstrated by terminal differentiation of hypodermal cells occurring one stage too early, during the L3 rather than L4 stage (Abrahante et al 1998; Jeon et al 1999; Tennessen et al 2006). In wild-type animals, lin-42 temporally restricts this differentiation event, at least in part, by acting as a negative transcriptional regulator of certain miRNA genes, including let-7 family miRNAs that have prominent roles in the heterochronic gene pathway (Reinhart et al 2000; Abbott et al 2005; Li et al 2005; McCulloch and Rougvie 2014; Perales et al 2014; Van Wynsberghe et al 2014). …”

Remodeling of theC. elegansNon-coding RNA Transcriptome by Heat Shock

“…Interestingly, the Period protein homolog lin-42 is strongly up-regulated in HS and a predicted target of four different miRNAs with reduced expression in HS (Figure 2E; Supplementary Table S2). This gene regulates molting cycles and acts as a general transcriptional repressor of miRNA genes (47)(48)(49)(50). Thus, increased expression of lin-42 in HS could contribute to transcriptional repression of many miRNA genes.…”