Does BspUPG-2 have a regulatory function?

We have shown that meiocyte production in apomictic Boechera is highly variable despite stable BspUPG-2 upregulation in generative tissues at the onset of meiosis and the formation of apomictic seeds (Figs. 9 and 15). The unexpected detection of a single candidate instead of multiple genes may therefore be interpreted in different ways with respect to its association with apomeiosis in anthers: either the locus controlling male apomeiosis contains a key genetic factor (e.g. BspUPG-2) with pleiotropic effects on other putatively essential components of unreduced pollen formation, or it contains several tightly linked factors, one of which is BspUPG-2, each controlling different aspects of unreduced pollen formation (i.e. apomeiosis). Therefore additional factors, including modifier genes (Bicknell et al., 2000), genetic background and/or environmental conditions (Nogler 1984) could explain variation in the level of apomictic trait expression.

The detection of the apo-specific indel9 is one example of a potential co-regulatory target site in BspUPG-2, although no homologous sRNAs were detected (Fig. 23B). Alternatively, the four detected mRNA isoforms (i.e. potentially caused by alternative splicing; Figs. 17 and 18) could regulate transcript abundance (as is predicted for at least 25% of all alternative exons; Stamm et al. (2005)) leading to the observed

variation in terms of reduced and unreduced pollen formation in both facultative and obligate apomicts.

The identification of an apomeiosis-associated short open reading-frame mRNA (sORF mRNA) and/or lncRNA is unique. Usually the identification of lncRNAs is difficult due to microarray designs, missing unannotated genes and the use of standard gene prediction programs that rely on the presence of relatively long ORFs (Zhang 2002). Here, we used detailed bioinformatic analyses of cDNA databases, including coding and miRNA formation potential in conjunction with chromosome walking to identify the chimeric BspUPG-2. Given its chimeric structure, the novel lncRNA BspUPG-2 could have attained neofunctionalization in the context of apomeiosis for pollen development through a number of mechanisms (Kaessmann 2010), similar to the lncRNAs BcMF11 from Brassica campestris (Song et al., 2007; 2012) or Zm401 in maize (Ma et al., 2008). In addition, its tissue-specific expression pattern and conservation at the nucleotide level strongly supports a developmental role for the lncRNA BspUPG-2. Hence, BspUPG-2 could conceivably have a regulatory function via a homology-dependent gene silencing mechanism (HDGS, reviewed in Meyer and Saedler (1996)), such as posttranscriptional gene silencing of related genes in trans (Eamens et al., 2008), which has been found for chimeric Helitron and Pack-MULE RNAs in maize (Jiang et al., 2004; Morgante et al., 2005), or via the modulation of DNA methylation patterns, such as reported for lncRNA-like loci which are associated with polycomb components and histone modifications (de Lucia and Dean 2011).

In line with this would be the observation of sequence fragments homologous to three different functional parental genes across the 5`-end of BspUPG-2 which putatively could serve as targets for HDGS (BspHRD3, RNAR and EFTU/EF-1A; Figs. 18 and 23). Thereby, BspUPG-2 could belong to a novel class of miRNA-containing lncRNAs which serve as matrices (i.e. as precursor RNA) for unidentified miRNAs or endogenous trans acting short interfering RNAs (ta-siRNA), as for example were detected in vegetative developmental pathways (Peragine et al., 2004; Vazquez et al., 2004; Hirsch et al., 2006) as well as in generative tissues (e.g. in mature pollen; Grant- Downton et al. (2009b)). Unlike other siRNAs in plants, ta-siRNAs silence gene expression by acting in trans to cleave mRNAs with sequences only partially complementary to their own (Peragine et al., 2004). The biogenesis of ta-siRNAs comprises their processing from the excised intronic region of a spliced transcript, or from the full-length unspliced transcript with Dicer-like proteins into 21 nt long siRNAs

which are incorporated into a RNA-induced Silencing Complex (RISC) with the appropriate Argonaute protein, which binds to target transcripts which are subsequently converted into dsRNA via RNA-dependent RNA polymerases (Peragine et al., 2004; Vazquez et al., 2004; Yoshikawa et al., 2005). One prominent example of this lncRNA- type is the pri-miR162a, whose miRNA product MIR162a putatively acts as substrates of DCL1 (Hirsch et al., 2006).

In search for secondary structures from BspUPG-2 similar to those mentioned above, database screens for known Boechera-specific and other plant sRNAs for homologous sequences were unfruitful. Despite these negative results, computer analysis of the RNA folding probability for different window slices (i.e. 50nt to 300nt with step size=10nt and window delta=10nt) of the complete gene using the ViennaRNA package (Hofacker et al., 1994) detected several sections of BspUPG-2 which form with high probability non-random and stable secondary structures (Fig. 19, Supplemental Figure 2 and Supplemental Tables 13 and 14). According to previous studies the minimal folding free energy index (MFEI) is an appropiate measure to distinguish miRNAs from other non-coding and coding RNAs, whereby 90% of miRNA precursors have a MFEI greater than 0.85 (Zhang et al., 2006). Interestingely, seven of the eight detected secondary structures have MFEIs greater than 1.07 and also fulfill other criterias for miRNAs (e.g. elevated A+U content, Supplemental Table 15). Surprisingly, these potential miRNAs were not detected in a previous screen (Amiteye et al., 2011), which might be due to their tissue-specific and short-term upregulation at the onset of male meiosis or due to shortcomings of previous analyses which often refer to homology-based screens, whereas BspUPG-2 demonstrated no homology to any known gene in close relative species.

In how far these conserved, structured and highly expressed RNA domains could be functional elements that play a role in posttranscriptional regulation of target genes in trans remains open as most of them demonstrated only minor homologies with known protein-coding genes. However, the detection of a stable secondary structure in a region which is highly homologous to a known protein-coding gene with translation elongation activities during polypeptide synthesis at the ribosome and activities in signal transduction (i.e. npcRNA 5 similar to GTP binding Elongation factor Tu/EF-1A family protein; E-value=7.00E-24, GO:0003746, AT4G02930), could be a first indication for a HDGS function of BspUPG-2. The highly conserved eukaryotic EFTU/EF-1A is involved in many cellular processes in plants, and hence modulation of EFTU/EF-1A

activity would have tremendous effects to the translation efficieny of many tRNAs, which it binds in a GTP-dependent reaction to the acceptor site of ribosomes (Fu et al., 2012). Interestingely, such modulation was observed for a homologous factor in Xenopus during meiotic progression, were some but not all subunits of EF-1 become phosphorylated by cdc2 kinase (i.e. amongst other kinases) - a metaphase promoting factor - during prophase to metaphase transition of meiotic cell division, resulting in an enhancernent of elongation activity (Bellé et al., 1990; Peters et al., 1995). It would now be of great interest to investigate if and how npcRNA 5 could be involved via interaction with EFTU/EF-1A in the regulation of protein synthesis during meiotic maturation of male gametes in Boechera. 

Noteworthy in this context is the observation that one of the other two sequence fragments of the 5`-end of BspUPG-2 is also homologous to a gene with nucleotide binding function (RNAR; GO:0003723), whereas the Arabidopsis homolog of BspHRD3 (AT1G18260) is involved in vesicle transport from the endoplasmic reticulum (ER) to the Golgi bodies (GO:0030433) and is associated with salt stress (GO:0042538). Expression of Arabidopsis homologs of all three parental genes was found across pollen development (Borges et al., 2008), and interestingly, greater abundance and development of endoplasmic reticulum, Golgi bodies, polysomes and mitochondrial cristae was found in unreduced compared to meiotically derived egg cells in mature aposporous embryo sacs of P. ciliare (Naumova and Vielle-Calzada 2001), an observation which was related to early egg cell maturation and the loss or truncation of the quiescent phase of egg cell development in apomicts. In this context an analysis of relative mRNA levels of the parental gene BspHRD3, which is involved in the ER to Golgi transport and which is actively transcribed in sexual and apomictic genotypes, would give further insights into its regulation and role during unreduced pollen formation in pseudogamous apomicts. Furthermore a functional analysis of the collection of BspUPG-2 npcRNAs should help us to better grasp the role of BspUPG-2 in unreduced pollen formation.

To summarize, despite variability for unreduced pollen formation in apomictic Boechera, a single novel transcription unit (BspUPG-2) is consistently upregulated in apomictic flower tissues at the PMC stage. BspUPG-2 has a chimeric sequence structure which might reflect the interspecific hybridization history of this genus. Whereas many studies have focused on mutation accumulation and deregulation with respect to origins of apomixis elements (e.g. Tucker et al. (2003), d’Erfurth et al.

(2008)), the emergence of novel genes in apomicts has not been appreciated, although various identified apomixis-associated loci suggest species-specific inheritance of this trait (Grossniklaus et al., 2001) for which “gains in function” are required (Vielle- Calzada et al., 1996). The identification of the novel apo-specific BspUPG-2 however, supports the HFA theory, which proposes that apomeiosis, and in a broader perspective apomixis, originates from hybrid-specific “genome collisions” and associated induction of gene duplication and TE activation (Carman 1997). How BspUPG-2 has undergone neofunctionalization to develop a hypothesized trans-regulatory function remains to be clarified.