5.3 PROPIEDADES ESTRUCTURALES
5.3.2 DEGRADACIÓN DE LA RIGIDEZ
The first proteins recognized to be m6A readers were those containing a YT521-B Homology (YTH) domain. Five of these proteins were identified in mammals, YTH domain containing (YTHDC) 1 and 2, YTH domain family (YTHDF 1-3) (Scutenaire et al., 2018). The YTHDF group of proteins consists of three extremely similar paralogs, yet different YTHDF proteins can perform distinct functions. Structural work performed on YTHDC1 demonstrated the YTH domain contained a hydrophobic core that accommodated the methyl-group in m6A and further that YTHDC1 demonstrated a sequence preference of GG(m6A)C, consistent with m6A’s preferred sequence context of RRACH, thereby demonstrating the importance of the sequence context of m6A in governing RNA-YTH domain interactions (Xu et al., 2014b, 2015). YTHDC1 is known to localize primarily within the nucleus (Nayler et al., 2000), whereas YTHDF1, YTHDF2, YTHDF3, and YTHDC2 are primarily cytoplasmic.
Consistent with its nuclear localization, YTHDC1 has been shown to regulate alternative splicing by promoting SRSF3 binding and exon inclusion in HEK293T cells. YTHDC1 is
necessary for proper localization of mRNA to nuclear speckles and was shown to directly compete with another splicing factor that promotes exon skipping: SRSF10 (Xiao et al., 2016). YTHDC1 was also shown to be important for nuclear export of m6A modified mRNA, as cross- linking and immunoprecipitation (CLIP) experiments identified binding targets of YTHDC1
accumulated in the nucleus while simultaneously being depleted in the cytoplasm when YTHDC1 expression was diminished. Nuclear export of these m6A transcripts is mediated by the
recruitment of SRSF3 by YTHDC1, as this study demonstrated only transcripts that were targets of both SRSF3 and YTHDC1 were enriched in the nucleus when YTHDC1 expression was abrogated (Roundtree et al., 2017a).
YTHDC2 is a primarily cytoplasmic m6A reader protein that is also known to be an RNA helicase. Studies in mouse spermatogenesis demonstrate that YTHDC2 is necessary for proper gamete development (Hsu et al., 2017) and progression through meiosis (Bailey et al., 2017;
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Wojtas et al., 2017). YTHDC2 interactions with the 5’ -> 3’ exoribonuclease XRN1 (Kretschmer et al., 2018; Wojtas et al., 2017) and also binds to rRNA in the small ribosomal subunit. Although rRNA contains m6A, the interaction of YTHDC2 with the ribosome was shown to be m6A
independent (Kretschmer et al., 2018). These interactions may help explain results from another study demonstrating that YTHDC2 interactions promote translation and destabilizes transcripts during spermatogenesis (Lin et al., 2017; Xu et al., 2017). The promotion of mRNA translation and destabilization may suggest a mechanism for tight regulation of transcripts necessary for cell differentiation programs that require timely transient expression of specific proteins for
progression to the next stage in differentiation.
Several questions remained after the discovery that YTHDC2 was an important factor in proper spermatogenesis. Is there an analogous requirement for m6A readers in female
gametogenesis? Furthermore, is the other YTHDC reader protein, YTHDC1, an important factor in gametogenesis? Another gap in knowledge stemmed from the fact that while the
spermatogenesis study demonstrated YTHDC2 was pivotal for proper gamete formation, it did not sufficiently demonstrate that this was due to the interaction of YTHDC2 with m6A. In this work, we attempt to fill in these gaps through investigation of the importance of YTHDC1 as an m6A reader throughout oocyte development.
YTHDF1 is known to promote mammalian translation. YTHDF1 binds m6A and interacts with translation machinery, namely EUKARYOTIC INITIATION FACTOR 3 (EIF3), to promote cap-dependent translation (Wang et al., 2015). Translatome experiments in YTHDF1 deficient cells also demonstrated that in the absence of YTHDF1, transcripts with either a known m6A or YTHDF1 binding site were, on average, significantly more ribosome associated and,
consequently, translated (Wang et al., 2015). Further analysis of transcripts with YTHDF1 PAR- CLIP sites demonstrated in the absence of m6A, translation efficiency was significantly lower compared to when m6A was present (Wang et al., 2015). Despite its similarity to YTHDF2, this mechanism appears to be unique to YTHDF1 (Wang et al., 2015).
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YTHDF2 is a cytoplasmically-localized protein that regulates transcript stability in an m6A dependent manner (Wang et al., 2014a). Similar to many of the other m6A readers, YTHDF2, is an important regulator of germline development. Oocyte maturation studies have shown that proper development cannot occur in the absence of YTHDF2 and that YTHDF2 deficiency is associated with misregulation of RNA metabolism (Ivanova et al., 2017). In this oocyte study, as well as experiments performed in HEK293T cells, mRNA targets of YTHDF2 exhibit increased stability in the absence of this RBP (Du et al., 2016; Ivanova et al., 2017).
This YTHDF2-mediated destabilization of transcripts in the HEK293T experiments was shown to be due to the recruitment of the CCR4-NOT deadenylase complex which subsequently degrades the polyA tail, destabilizing the transcripts (Du et al., 2016). Likewise, cancer cells lacking YTHDF2 demonstrated upregulation of SOCS2 expression, potentially due to increased stability in the absence of YTHDF2 binding (Chen et al., 2018). In mouse neurons, however, reports suggest YTHDF2 destabilizes transcripts through recruitment of the FRAGILE-X MENTAL RETARDATION PROTEIN, indicating that the regulation of transcript stability may be heavily dependent on context (Zhang et al., 2018).
YTHDF3 has been shown to be important for both RNA degradation (Shi et al., 2017) and translation (Li et al., 2017; Shi et al., 2017). PAR-CLIP experiments demonstrate a high amount of overlap between YTHDF1, 2, and 3, and co-IP experiments show that these three YTH proteins all interact (Shi et al., 2017). Evidence demonstrates YTHDF3 may be the first reader protein to bind a cytoplasmic transcript where models suggest it can then enhance the transcript’s interaction with YTHDF1 and therefore translation of the transcript. In the absence of YTHDF3, YTHDF1-bound transcripts are less well translated (Shi et al., 2017). However, the possibility remains that YTHDF3 is not facilitating YTHDF1 and 2 binding, but it may perform redundant functions and facilitate translation on its own. Further experiments are required to determine whether YTHDF3 functions independently or in concert with YTHDF1 and 2.
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While great strides have been made in characterizing the YTH m6A reader proteins in mammals, less is well known about this protein family in plants. In Arabidopsis,a substantial expansion in the proteins containing a YTH domain has occurred. In fact, 13 proteins contain a YTH domain, consisting of two YTHDC1 homologues and 11 members in the YTHDF clade (Arribas-Hernández et al., 2018; Scutenaire et al., 2018). Despite the large number of potential m6A readers in plants, only three have been studied in the context of m6A to date:
EVOLUTIONARILY CONSERVED C-TERMINAL REGION 2, 3, and 4 (ECT2, ECT3, ECT4). ECT2-4 are members of the YTHDF clade of m6A readers; ECT2 and 3 are the most highly and widely expressed of the putative m6A reader proteins in Arabidopsis (Arribas-Hernández et al., 2018).
ECT2 is ubiquitously expressed throughout various organs and developmental timepoints in Arabidopsis, with the highest level of expression observed in seeds (Scutenaire et al., 2018) and other rapidly developing tissues (Arribas-Hernández et al., 2018; Wei et al., 2018b). The localization pattern of ECT2 remains unclear as ECT2 localization findings differ between studies. Specifically, two studies concluded ECT2 is largely cytoplasmic (Arribas-Hernández et al., 2018; Scutenaire et al., 2018), whereas a different study reports ECT2 localizes to both the cytoplasm and nucleus (Wei et al., 2018b).
In plants deficient in ECT2, aberrant trichome morphology was observed, with nearly 50% of trichomes from ECT2 deficient plants exhibiting 4 or more branches (Scutenaire et al., 2018; Wei et al., 2018b). Trichomes with these higher levels of branching exhibited evidence of higher ploidy levels (Scutenaire et al., 2018), indicating that excessive branching was a result of excessive endoreduplication. Similar trichome morphology was observed in plants deficient in m6A writer proteins (Bodi et al., 2012a), and ECT2 binding to m6A was demonstrated to be necessary for proper trichome morphology through mutations in the m6A binding domain (Scutenaire et al., 2018). Furthermore, ECT2 under heat stress conditions localized primarily to stress granules, demonstrating that ECT2 and its interaction with m6A is likely important for both
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development and stress response (Scutenaire et al., 2018). In contrast to most reports involving YTH proteins in mammals, ECT2 has a stabilizing effect on transcripts (Wei et al., 2018b).
ECT3 works in concert with ECT2 and ECT4 to control developmental programs. Specifically, while plants deficient in one of the ECT2-4 proteins showed no visible defect with leaf formation, ect2/ect3 double mutants exhibited delayed leaf formation and ect2/ect3/ect4 triple mutants exaggerated this delay. All three of these proteins demonstrated high localization to sites of leaf formation (Arribas-Hernández et al., 2018). Furthermore, ect2/ect3 double and
ect2/ect3/ect4 triple mutants exhibited a significantly reduced leaf size and number relative to wild-type plants 27 days after germination. Importantly, these morphology changes were observed when the m6A binding domain in these proteins was mutated to prevent m6A
interaction, demonstrating that these developmental programs are a result of m6A recognition and binding (Arribas-Hernández et al., 2018).