Long and short isoforms of Neurospora clock protein FRQ support temperature-compensated circadian rhythms

Abstract

The large (l) and small (s) isoforms of FREQUENCY (FRQ) are elements of interconnected feedback loops of the Neurospora circadian clock. The expression ratio of l-FRQ vs. s-FRQ is regulated by thermosensitive splicing of an intron containing the initiation codon for l-FRQ. We show that this splicing is dependent on light and temperature and displays a circadian rhythm. Strains expressing only l-FRQ or s-FRQ support short and long temperature-compensated circadian rhythms, respectively. The thermosensitive expression ratio of FRQ isoforms influences period length in wt. Our data indicate that differential expression of FRQ isoforms is not required for temperature compensation but rather provides a means to fine-tune period length in response to ambient temperature.

Fig. 1

Splicing efficiency of frq-I6 is set by temperature and modulated in a circadian and temperature dependent fashion. Neurospora cultures were grown at the indicated temperatures and harvested (A) in constant light (LL) and (B) after the indicated time periods in darkness (DD). RNA was prepared and quantified by RT-PCR using fluorescent probes specific to spliced frqI6 and to total frq RNA [14]. The fraction (in %) of frq RNA spliced at I6 (spliced/total frq RNA) was plotted versus the time in DD. Black and white bars indicate subjective night and day, respectively.

Fig. 2

l-FRQ to s-FRQ determines the length of free running period. (A) Western blot analysis of protein extracts from cultures grown in LL at the indicated temperatures. Protein extracts were treated with phosphatase prior to SDS PAGE. Optimizing the splice sites of intron 6 (s-frq) results in the expression of s-FRQ while mutation of the splice sequences (l-frq) results in expression of l-FRQ. The temperature dependence of expression levels of FRQ is not affected by mutation of frq-I6. (B) Typical racetubes of s-frq, l-frq and the corresponding wt (wt^cont.) at 22°C and at 28°C. Conidial bands are indicated by arrow heads and numbered. The growth rate of the strains was identical. (C) l-frq and s-frq support considerably well temperature compensated short and long period rhythms, respectively. The period lengt of wt is between that l-frq and s-frq. Data represent assessment of the temperature dependence of the free-running period of l-frq, s-frq, and wt^cont. from 183 racetubes, each with an average of 5 conidial bands (see also Table 1A). The dotted line represents a hypothetical uncompensated reaction with a Q₁₀ of 2.

Fig. 3

frq-ATG1^mut expresses predominantly s-FRQ and supports a long period circadian rhythm. (A) Schematic outline of the translation initiation sites of FRQ isoforms and intron 6. Top: wt donor, lariat (bold italic) and acceptor sites are shown above the outline. The large asterisk indicates the sequenced acceptor site, the small asterisks indicate putative cryptic acceptors. Corresponding sequences mutated in hvc-16 and l-frq are also shown, sequence alterations are underlined. Bottom: Sequence context around the initiation sites of l-FRQ, l*-FRQ and s-FRQ. Numbers below the sequence refer to codons of l-FRQ. The initiation ATGs of l*-FRQ and s-FRQ correspond to codons 11 and 100 of l-FRQ, respectively. The sequence alterations introduced in the frq-ATG1^mut allele are shown. (B) Western blot analysis of FRQ levels and isoforms expressed in frq-ATG1^mut at the indicated temperatures in constant light. (C) Racetube assays revealing that the free-running periods of frq-ATG1^mut and s-frq are similar (see also Table 1A).

Fig. 4

(A) l- and s-FRQ expressed in hvc-16 in constant light. Western blot analysis of protein extracts from cultures grown in LL at the indicated temperatures. Protein extracts were treated with phosphatase prior to SDS PAGE. At 15 and 25°C low levels of s-FRQ can still be detected in the donor mutant hvc-16. (B) Racetube assay revealing that the free-running periods of hvc16 (21.7) and wt^cont (21.5) are similar at 25°C (n=6). (C) Evolutionary conservation of s-FRQ in filamentous fungi. Alignment of the N-terminal part of the amino acid sequence of FRQ proteins of N.c., Neurospora crassa; S.f., Sordaria fimicola; L.a., Leptosphaeria australiensis; H.s., Hypocrea spinulosa. Methionine residues (M) are printed in bold.