El príncipe Dionís es coronado como rey de Polonia

3.1 DESCRIPTION OF THE PAPERS!

In this chapter I will outline aims and results of the four papers I contributed to during my PhD course. In Paper 1 we performed a thorough comparative investigation of the ORFans (fORF and mORF) found in the mt genomes of bivalves with DUI, to gain insights into their origin and function. We studied the degree of conservation of these ORFans in different species and investigated the functions of the putative proteins encoded by them. My role was to search for ORFans presence and conservation in all the available mt genome sequences of known DUI species in GenBank, plus Ruditapes philippinarum and Musculista senhousia mtDNA segments specifically sequenced by me and the research group. Paper 2 aim was to understand why bivalve mt genomes retain unusually large unassigned regions, and to understand how F and M mtDNA are transcribed in Ruditapes philippinarum. We described these regions and analyzed the transcriptome data from female and male gonads, also obtaining data on sequence variability of the two mt genomes. My contribution to this work was to sequence and annotate the largest unassigned regions of Ruditapes philippinarum mt genomes, to identify features (such as elements typical of a CR or ORFans) that could justify their retention. For Paper 3 I contributed as first author in the characterization of the largest URs of Musculista senhousia F and M mt genomes. I thoroughly annotated these regions, and suggested that they contain the CR of the respective mt genomes. The CR is duplicated in the F mtDNA: by examining the conservation of the two copies through a Bayesian analysis, I provided evidence for their concerted evolution. Paper 4 is a review that

summarizes the current knowledge on the unusual features found in many animal mt genomes, and in particular about abnormal gene content. Additional genes or ORFans not related to the OXPHOS system may greatly expand the role of mitochondria in an organism, even in processes such as sex determination or speciation. For this review, I was in charge of the section on gene duplications. The following summaries are intended to give a general overview of the papers, the topic they investigate, the methods used and the results they brought. A brief discussion contextualizing the findings closes every summary. Being Paper 4 a review, no such sections are reported in its summary. Author affiliations are enlisted at the end of this chapter. Please also read the full papers attached in Appendix 2 to have a more detailed view of the works.

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Figure 16 Schematic structure of Musculista senhousia largest URs in its F and M mt genomes.

FUR2 contains the fORF, while Subunits B in the LURs contain ORF-B on the reverse strand. ORF-B is sometimes disrupted in the F mtDNA. See Figures 8 and 9 for the location of these regions in the respective mtDNA. [Taken from Milani et al. (2013)]

Figure 17 Detailed structure of Ruditapes philippinarum F and M mtDNAs largest unassigned regions. MUR21 contains the mORF, while the fORF is comprised in FLUR. RS: RNA secondary structures, DS: DNA secondary structures. See Figures 6 and 7 for the location of these URs in their respective mt genomes. [Modified from Ghiselli et al. (2013)]

3.2 PAPER 1

A comparative analysis of mitochondrial ORFans: new clues on their origin and role in species with doubly uniparental inheritance of mitochondria!

Liliana Milani¹, Fabrizio Ghiselli¹, Davide Guerra¹, Sophie Breton², Marco Passamonti¹! Genome Biology and Evolution 2013, 5 (7): 1408-1434!

3.2.1 BACKGROUND!

Additional ORFs are being continuously found in animal mt genomes. Some of them have been shown to derive from duplication and divergence of extant genes, as in the bivalves Ruditapes philippinarum (Okazaki and Ueshima, unpublished data; Figure 6 in Chapter 1), Musculista senhousia (Passamonti et al. 2011; Figure 9 in Chapter 1), and in the genus Crassostrea (Wu et al. 2012a), or in some hydroidolinan hydrozoans (Kayal and Lavrov 2008, Voigt et al. 2008). Additional genes found in Cnidaria have been hypothesized to derive from horizontal transfers (from linear plasmids, ε-proteobacteria, or viruses; Claverie et al. 2009, Bilewitch and Degnan 2011, Ogata et al. 2011, Kayal et al. 2012).!

In DUI species belonging to the orders Mytiloida, Unionoida, and Veneroida, both F and M mtDNAs contain additional lineage-specific ORFans, respectively named fORF and mORF (Breton et al. 2009, Breton et al. 2011a, Breton et al. 2011b, Ghiselli et al. 2013):

these ORFans are usually located in large URs or in the CR. In the order Mytiloida, both fORF and mORF of Mytilus spp. are situated in the CR first variable domain (named VD1, see Figure 10 in Chapter 1; Cao et al. 2004b) of the respective mtDNAs (Breton et al. 2011b), and a fORF is present in UR2 of Musculista senhousia F mtDNA (Figure 8 in Chapter 1, Figure 16; Passamonti et al. 2011, Breton et al. 2011b). In Ruditapes philippinarum, fORF and mORF are located respectively in the FLUR and in MUR21 of the respective mtDNAs

(upstream nad4L in both mt genomes, see Figures 6 and 7 in Chapter 1, Figure 17; Ghiselli et al. 2013). Finally, in Unionoids, fORF is located upstream nad2 in the F mtDNA (except for Inversidens japanensis and Hyriopsis cumingii, which have a different gene order), while mORF is placed upstream nad4L in the M (Breton et al. 2009).

These ORFans have been hypothesized to be linked to the maintenance of gonochorism in these species, as well as to be responsible of the peculiar mitochondrial transmission system that is DUI (Breton et al. 2009, Breton et al. 2011a, Breton et al. 2011b). The finding that the two lineage-specific ORFans are translated in the unionid Venustaconcha ellipsiformis (Breton et al. 2009), and in particular that the fORF protein product is present in mitochondria, nuclear membrane, and egg nucleoplasm of this species (Breton et al. 2011a), supports their direct involvement in the DUI mechanism.!

For this work we sequenced and analyzed the major mt URs of the DUI Mytilid Musculista senhousia to search for novel ORFans, and compared them with those found in other DUI species. A functional analysis on the proteins produced by these ORFs was performed to gain insights into their function, origin, and putative role in the DUI system.!

3.2.2 ANALYSES AND RESULTS!

We sequenced the LURs of Musculista senhousia F and M mtDNA plus FUR2 to confirm the presence of fORF and search for other, not previously annotated, additional ORFs, using DNA extracted from 12 egg samples and 11 sperm samples spawned from different specimens. fORF presence was confirmed in all analyzed sequences and a new ORF on the reverse strand, named ORF-B, was found in both FLUR and MLUR (Figure 16):

FLUR is composed of two large tandem repeats (Passamonti et al. 2011) and this ORF is found in both of them. ORF-B is always conserved in males, while in some female specimen

its sequence showed mutations and deletions that altered the reading frame, modifying its length or breaking it in two overlapping ORFs.!

I gathered all complete and partial CR sequences of the genus Mytilus from GenBank to confirm the presence of fORF and mORF in the VD1 described by Breton et al. (2011b). 689 sequences belonging to four species of Mytilus (Mytilus edulis, Mytilus galloprovincialis, Mytilus californianus, Mytilus coruscus) were analyzed: 201 of them contained full-length ORFs (197 fORFs and 17 mORFs; some sequences are recombinant and can contain more than one ORF). All cases where fORF was incomplete were just because the VD1 was not fully sequenced, thus truncating the fORF sequence: the reading frame of all partial and full fORFs was found intact. On the contrary, most of mORF showed a disrupted reading frame.

The mORFs annotated by Breton et al. (2011b) in Mytilus edulis, Mytilus galloprovincialis, and Mytilus trossulus, comprised a long poly-A string in their reading frame: the different number of adenines in this string, and mutations upstream of it, were the main reasons why many mORFs were found compromised. Nonetheless, the part downstream this homopolymers is conserved in the majority of mORFs.

I then assessed the variability and conservation of all available fORF and mORF sequences from the DUI species Musculista senhousia (obtained in this study; also ORF-Bs were considered), the four Mytilus species (from GenBank), Ruditapes philippinarum (obtained by Ghiselli et al. 2013), and Venustaconcha ellipsiformis (from GenBank), using p-distance (Table 3). In Musculista senhousia fORFs are more variable than male ORF-B, but more conserved than female ORF-B. Mytilus fORFs are less variable than mORFs, while in Ruditapes philippinarum mORF is more conserved than fORF. All available Venustaconcha ellipsiformis fORF sequences are identical among each other. I calculated the p-distances also for the translation of the ORFs (Table 3): all values are higher than those of the respective nucleotide sequences, except for Ruditapes philippinarum mORF where the p-D value is zero.

Table 3 p-Distance (p-D) and Standard Error (SE) values of novel mitochondrial ORFs in DUI bivalves. Number of ORF sequences used for each species is dependant on the number of available and suitable sequences on GenBank. p-D of Mytilus edulis, Mytilus galloprovincialis, and Mytilus trossulus mORFs were calculated only on the last part of the ORF immediately following the poly-A sequence (see Milani et al. 2013 in Appendix 2 for details). N = number of sequences used. ^a Only complete female ORF-B were considered. ^b Male ORF-B and complete female ORF-B were considered. ^c mORF sequences matching Mytilus edulis mORF. [Taken from Milani et al. (2013)]

The translations of the lineage-specific ORFans of DUI species (henceforth named FORF and MORF), Musculista senhousia ORF-B, an ORF found in Paphia euglypta (Bivalvia Veneridae) CR, and the additional ORFs in three cnidarian species, were analyzed to infer their structure and used for a function prediction analysis. A SP was found at the N-terminus of all FORFs, always coincident with a transmembrane helix; the same situation was found in MORFs, even if the SP support was lower. The function prediction for FORFs gave many hits with proteins involved in nucleic acid binding, transcription, RNA modification or methylation, membrane association, and immune response. For MORFs, the hits were related to proteins having a role in membrane association with nucleic acid binding and transcription, DNA recombination, transcription, integration of foreign elements, cytoskeleton dynamics, ubiquitination, apoptosis, and immune response. For all DUI bivalves lineage-specific

ORFans, the first four hits found by one software were always the same: two hits are involved in cell membrane/surface anchoring, and the other two to transcription and post-transcriptional processes. Finally, all analyzed ORFs showed hits with viral proteins, except for the MORFs of Mytilus trossulus and Venustaconcha ellipsiformis. See Figure 18 for location of the hits on the protein sequences.!

3.2.3 DISCUSSION!

The conservation in DUI species of most of all analyzed additional ORFans points to a functional meaning of these sequences. Exceptions are Musculista senhousia ORF-B, and Mytilus edulis, Mytilus galloprovincialis, and Mytilus trossulus mORFs. ORF-B is not lineage-specific and its sequence is compromised in many F mtDNA samples: this may indicate that this ORF is not functional in this mt genome. The mORF of the three Mytilus species mentioned above contain, in their “full length”! version, a long string of adenines (Breton et al. 2011b), and since most reading frames are compromised in this segment or in the part upstream of it, this may reflect a difficulty in sequencing this long homopolymer. All other lineage-specific ORFans in DUI species are conserved, with variable patterns among species.!

The structure of the putative proteins translated from the ORFans suggests an anchoring to membranes, in particular for MORFs of Mytilus and Ruditapes philippinarum. All proteins share similar functions, but when only considering most supported hits, FORFs are more similar among each other than with MORFs, and vice versa. Both FORFs and MORFs have hits indicating a role as signaling molecules, but most functions are typical of only one ORFan product type (FORFs: transcription regulation and immune response, cell adhesion, migration, and proliferation; MORFs: cytoskeleton organization, cell differentiation during embryonic development, nucleic acid binding and transcription regulation). Some MORFs

have similarities with DNA replication, recombination, and repair proteins, and almost all ORFan proteins show hits of ubiquitination and apoptosis regulation proteins.!

Given the results, it is unlikely that the lineage-specific ORFans analyzed originated from duplication of standard mtDNA-encoded genes. The high amino acid substitution rate observed (compared to typical mt genes) could be the signature of lineage-specific adaptation (Daubin and Ochman 2004a, Daubin and Ochman 2004b, Cai and Petrov 2010, Yu and Stoltzfus 2012), and since many hits refer to viral proteins, this may indicate a viral origin of these ORFans. These viral sequences might have been endogenized by the host organism and co-opted for host cell functions (Feschotte and Gilbert 2012), and clues coming from the structural and functional analyses open many possibilities. For example, these foreign elements could originally have a role in immune response: since mitochondria have central roles in regulating immune response, the viruses from which these ORFans could come from might have targeted mitochondria to evade this process (Ohta and Nishiyama 2011); also, some structural features also suggest involvements in apoptosis control. Viral envelope proteins have been shown to cause aggregation of mitochondria (Doorbar et al. 1991, Galluzzi et al. 2008), and the many MORFs hits concerning cytoskeleton can be related to a role in the differential distribution of sperm mitochondria during male and female development. Retrograde signaling from mitochondria to nucleus has been demonstrated in plants showing CMS (Abad et al. 1995, Fujii and Toriyama 2008, Nizampatnam et al. 2009), a system of sex determination involving additional mtDNA-encoded proteins that may bind to mitochondrial membranes (Nizampatnam et al. 2009). The novel proteins identified in DUI bivalves can putatively tag the outer membrane of mitochondria: in particular, MORFs might mask sperm mitochondria from the degradation machinery in developing male embryos.

Finally, these proteins can be located outside mitochondria (Breton et al. 2011a), contributing to a communication system between mitochondria and nucleus.!

The co-option of the novel ORFans in bivalve mtDNAs may have influenced some aspects of their life cycle involving mitochondria (Forterre 2006, Koonin 2006). In particular, selfish viral sequences might have found a way to be transmitted through the generations, leading to DUI. DUI presence is scattered in bivalves and the findings of our work may support a multiple origin of this mechanism. On the other hand, the similarities among ORFans may suggest an origin from elements of the same kind, but their conservation only among related taxa may indicate either independent origins or that their fast evolution has cancelled all sequence similarities.!

Figure 18 (next page) Functional domains in FORFs and MORFs of DUI species mtDNAs (position in the amino acid sequence as identified by HHpred; Söding et al. 2005). Sequences with similarities are boxed in the same color and with the same type of line; red: similarities among FORFs; blue:

similarities among MORFs; orange: K = poly-K region, S = poly-S region. Numbers indicate aa length.

[Taken from Milani et al. (2013)]

3.3 PAPER 2!

Structure, transcription, and variability of metazoan mitochondrial genome:

perspectives from an unusual mitochondrial inheritance system!

Fabrizio Ghiselli¹, Liliana Milani¹, Davide Guerra¹, Peter L. Chang³, Sophie Breton², Sergey V. Nuzhdin³, Marco Passamonti¹!

Genome Biology and Evolution 2013, 5 (8): 1535-1554!

3.3.1 BACKGROUND!

The protomitochondrion (Müller and Martin 1999, Atteia et al. 2009, Abhishek et al.

2011, Thrash et al. 2011) genome underwent a massive process of GRE since the ancestral symbiosis event (Andersson and Kurland 1998, Khachane et al. 2007), which transformed it into the mt genomes we know today in the various eukaryote lineages, shaped by both neutral and adaptive modifications (Embley and Martin 2006). Selective pressure for GRE (thus for UR deletion) is stronger in genomes with a high mutation rate, as non-functional intergenic DNA can accumulate gain-of-function harmful mutations (Lynch et al. 2006, Lynch et al.

2011, Lynch 2007), but its efficiency is dependent on the amount of random genetic drift and effective population size.!

mtDNA mutation rate is variable among animal lineages, and it has been linked to body mass, metabolic rate, ROS production, and lifespan (Galtier et al. 2009). Most of the variability however seems due to errors made by DNA polymerase during replication (Drake et al. 1998, Lynch et al. 2006): it follows that large part of the heritable mutations are accumulated during germ line proliferation, where cells undergo a great number of divisions.

Thus, reproduction modes and gonad physiology may affect mtDNA rates of evolution (Rand 2001, Davison 2006). In bivalves, the high number of cell divisions in both female and male

gonads (which are produced de novo every reproductive season; Gosling 2003), and the large number of gametes produced, lead to a high mutation rate: indeed, in both nuclear and mt genomes, bivalves show a large amount of polymorphism (Saavedra and Bachere 2006). In addition, their mt genomes are very long, show an extremely variable gene order, and can be transmitted both maternally and paternally under DUI.!

The main aims of this work were to identify the reasons why large URs can be conserved in bivalve mt genomes, and test the transcription of F and M mtDNA in a DUI species, also analyzing the amount of polymorphism of the two mtDNAs. For this, we sequenced the largest URs of Ruditapes philippinarum F and M mtDNAs to locate the CR and identify other features that could explain the presence of such large URs. Then, we characterized the mitochondrial transcriptome of female and male gonads of this species, and performed a SNP analysis.!

Table 4 Proportion of URs in the mitochondrial genomes of Metazoans. N: sample number, median total length: median total length of the mt genomes in a taxon, median URs length: median total length of mtDNA URs in a taxa, median %cod: median proportion of coding regions in the mt genomes, median %URs: median proportion of URs in the genomes. Significance: Wilcoxon rank-sum test significance (***: P<0.001, n.s.: nonsignificant). [Taken from Ghiselli et al. (2013)]

3.3.2 ANALYSES AND RESULTS!

2,656 complete animal mt genomes were downloaded from the MitoZoa database to analyze the length and proportion of UR in various taxa. Bivalves are the group showing the

I used the DNA extracted from sperms and eggs spawned by 7 males and 8 females, respectively, of Ruditapes philippinarum as a template to amplify the mtDNA regions containing LUR and UR21 of the M and F mt genomes (the largest URs of the two mtDNAs;

see Figures 6 and 7 in Chapter 1). The obtained URs were analyzed in detail to search for conserved domains and secondary structures (Figure 17). Three main subunits, named A, B, and C, were found conserved among the URs, though their position is different between F and M mtDNA. A region containing a variable number of tandem repeats was found in the FLUR.

Some of the identified DNA secondary structures share similar loops in both F and M mtDNAs, and three RNA structures are conserved between the two mt genomes. In both mt genomes, in the same relative position (upstream the nad4L gene), an additional ORF was found conserved (fORF in the F and mORF in the M mtDNA): these ORFs have been analyzed in detail in Milani et al. (2013).!

To locate OH and OL in Ruditapes philippinarum mtDNAs, I performed an AT-skew analysis on its complete F and M mt genomes available in GenBank, and of other Veneridae species for additional support. In general, OH seems to be located in coincidence of the largest URs in all mt genomes, while the OL is often associated to a conserved cluster of three tRNA genes (tRNA-His, tRNA-Glu, and tRNA-Ser). I also searched the large URs of all these species for motifs typical of a CR by comparing them with the CR of the sea urchin Strongylocentrotus purpuratus, in which mtDNA replication is well characterized: two motifs (named δ! and γ; Figure 17) in Ruditapes philippinarum FUR21 and MLUR showed similarities with the motifs involved in the start of replication in sea urchin mtDNA.!

To perform a transcriptome analysis of the mt genomes from 6 male and 6 female gonads of Ruditapes philippinarum, a cDNA library was sequenced with an Illumina GAIIx platform. The majority of transcripts (90.11%) in male gonads are from M mtDNA. F mt genome transcription profiles are the same in males and females, and are different from that

of the M. mORF transcription levels are comparable to those of the other M genes, while fORF levels are significantly lower than all other F genes. A SNP analysis was performed on the mt transcriptomes to assess the variability of F and M mt genomes. F and M have similar amounts of high-frequency alleles, but the F mt genome, in both sexes, has an excess of rare

of the M. mORF transcription levels are comparable to those of the other M genes, while fORF levels are significantly lower than all other F genes. A SNP analysis was performed on the mt transcriptomes to assess the variability of F and M mt genomes. F and M have similar amounts of high-frequency alleles, but the F mt genome, in both sexes, has an excess of rare