Tendencia Otaku

Both the SR and hnRNP proteins have been shown to undergo AS, with NMD coupled to AS occurring in both protein families indicating that this may be a mechanism of fine-tuning the levels of these trans acting splicing factors.

1.3.2.1 SR proteins

Expression levels of SR proteins are differentially expressed in different organisms and developmental stages indicating that this may be one mechanism for organ- specific AS in plants due to the fact that SR factors regulate alternative splicing (Palusa et al., 2007). With over 88.9% of SR proteins undergo AS in Arabidopsis implying that they regulate and are regulated by AS mechanisms (Chamala et al., 2015; Richardson et al., 2011). Overexpression of atSR30 in transgenic Arabidopsis plants alters the splicing patterns of a variety of endogenous plant genes, including

atSR30 itself, as well as down-regulating the mRNA encoding the full-length

atSR34/SR1 protein, an SR protein with which it shares strong sequence similarities, with both these genes are deemed to encode SF2/ASF-like proteinsdue to their strong sequence similarities to human SF2 (Lopato et al., 1999). The splicing patterns of the mRNA encoding the SF2/ASF-like proteins atSR30 and atSR34 are altered in transgenic Arabidopsis plants overexpressing the plant specific SR protein atRSZ33

(Kalyna et al., 2003),indicating that SR proteins can regulate AS of their own genes as well as other endogenous plant genes. Further evidence that SR proteins can modulate splicing in plants comes from Isshiki et al (2006) work in rice protoplasts where they showed that the SR proteins RSp29 and RSZ23 enhanced splicing and altered the A5SS of the first intron of the Waxyb genes; RSp29, a plant specific SR protein, and RSZ23, an SR protein homologous to human 9G8, enhanced splicing at the distal and proximal site respectively. In transgenic rice plants over expressing the

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plant specific SR protein RSZ36 or the rice homolog of human SF2/ASF, SRp33b, alters the splicing patterns of their own pre-mRNAs and those of other SR proteins (Isshiki et al., 2006). Taken together these findings indicate that both plant specific SR proteins, and those with mammalian homologues, play a role in modulating splicing by regulating the mRNA splicing of plant genes via auto regulation and regulation of endogenous plant genes (including those that encode other SR genes proteins).

SR proteins have been shown to be regulated by AS coupled to NMD (Kalyna et al., 2012), in humans AS coupled to NMD of SR proteins is frequently used as an auto regulation mechanism and this may also be the case in plants. Paulsa et al 2010 found that in Arabidopsis mutants lacking upf3, one of the core components of the NMD pathway, there is a several fold increase in the transcript of SR30 (Palusa & Reddy, 2010). As mentioned above SR30 is found to be auto regulated, thus AS coupled to NMD may be one mechanism by which SR protein levels are fine-tuned. However not all SR transcripts containing PTC accumulated in the upf3 mutants plants indicating that they may produce truncated proteins; given the fact that SR proteins have multiple functional domains these transcripts may produce protein with an altered function (Palusa & Reddy, 2010).

The splicing patterns of SR proteins are also thought to play a functional role in the plant stress responses. SR proteins expression levels are not dramatically altered in response to stress (Duque, 2011), however SR proteins are extensively DAS in

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Table 2. SR genes known to undergo stress-mediated DAS.

1.3.2.2 hnRNP

Of the Arabidopsis hnRNP proteins with human homologues, a superfamily of 21 glycine-rich RNA-binding proteins (RBPs) that were homologs of human hnRNP A1 and hnRNP A2/B1 was identified; this family can be divided into two subfamilies

Gene

Identifier Name DAS under stress Reference

At1g02840 SR34/SR1 Temperature, hormones, _{heat, and cold} ( Lazar & Goodman, 2000; Palusa _{al., 2007)} et

At1g09140 SR30 Salt, _heat. Pst, high light, and (Ding 2010; Howard et al., 2014; Filichkin et al., 2013; Palusa et al., et al., 2007)

At1g16610 SR45 Low temperature, heat, _{and drought} (Gulledge₂₀₀₇₎ et al., 2012; Tanabe et al.,

At1g23860 RSZ21/SRZ-21 - -

At1g55310 SCL33 Salt, hormones, salt, cold, _{heat and glucose} (Ding ₂₀₀₇₎ et al., 2014; Palusa et al.,

At2g24590 RSZ22a - -

At2g37340 RSZ33 Salt, heat, and glucose (Ding ₂₀₀₇₎ et al., 2014; Palusa et al.,

At2g46610 RS31A Heat (Palusa et al., 2007)

At3g13570 SCL30a Salt and heat (Ding ₂₀₀₇₎ et al., 2014; Palusa et al.,

At3g49430 SR34a Salt (Ding et al., 2014)

At3g53500 RSZ32 Salt and heat (Ding ₂₀₀₇₎ et al., 2014; Palusa et al.,

At3g55460 SCL30 Salt (Ding et al., 2014)

At3g61860 RS31 Salt and cold (Ding ₂₀₀₇₎ et al., 2014; Palusa et al.,

At4g02430 SR34b Heat, cold, hormones, _{and salt} (Palusa et al., 2007)

At4g25500 RS40/RSP35 Salt, cold and heat (Ding ₂₀₀₇₎ et al., 2014; Palusa et al.,

At4g31580 RSZ22/SRZ-22 - -

At5g18810 SCL28 - -

At5g52040 RS41 Salt and heat (Ding et al., 2014; Palusa et al.,

2007)

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based on the number of RRM domains, 8 proteins contain one RRM domain and the remaining 13 contain two RRM domains(B.-B. Wang & Brendel, 2004b). Of these RBPs with one RRM domain AtGRP7 and its paralog AtGRP8 have been shown to regulate AS of pre-mRNA in an auto and cross regulatory manner (Schöning et al., 2008; Staiger et al., 2003). This regulation is thought to occur via AS coupled to NMD; AS transcripts of AtGRP7 and AtGRP8 contain PTCs, in NMD-impaired mutant plants and under cyclohexamide treatment accumulation of these mRNAs transcripts occurs indicating that those transcripts with the PTC are targets for NMD (Schöning

et al., 2007; 2008; Staiger et al., 2003). Transgenic plants that have a point mutation

of a conserved arginine to glutamine within the RNA recognition motif of AtGRP7

(AtGRP7-RQ-ox) display a reduction in its binding affinity, which disrupts the auto-

regulation of AtGRP7 and the regulation of downstream targets (Schöning et al., 2007). Streitner et al (2012) utilized a HR RT–PCR-based AS panel to determined that 20% of analysed AS events displayed changes in the ratios of AS isoforms under ectopic AtGRP7 expression and approximately half of these were also altered by the paralog AtGRP8, indicating that they may the share downstream targets. Several of the transcripts with altered AS ratios were deemed to be bound by AtGRP7 in vivo, plus the altered AS ratio of these transcripts did not occur in AtGRP7-RQ-ox mutant transgenic lines indicating that AtGRP7 affects the AS of these transcripts by directly interacting with them (Streitner et al., 2012). AtGRP7 is rhythmically activated by the endogenous circadian clock (Staiger & Apel, 1999), with global profiling of transgenic plants that constitutively overexpressed AtGRP7 (AtGRP7-ox) showing enrichment of genes controlled by the biological clock (Streitner, Hennig, Korneli, & Staiger, 2010). Transcripts responsive to abiotic and biotic stress were enriched in AtGRP7-ox transgenic lines, with transcripts encoding two of the pathogenesis-related proteins (PR1 and PR2) being elevated in AtGRP7-ox plants but not in AtGRP7-RQ-ox

transgenic plants, indicating that at least for some of the enriched transcripts direct binding of AtGRP7 is involved in their regulation (Streitner et al., 2010). Further evidence of the involvement of AtGRP7 in plant immunity was demonstrated by Fu

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susceptible to Pseudomonassyringae pv. tomato (Pst) than wild-type plants. These experiments taken together indicated that some hnRNPs are involved in the plant defence response by regulating AS of defence-related genes, potentially via direct binding of the pre-mRNA.