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\begin{document}

\title{Polyadenylation-based mitotranscriptomics of Apis mellifera (Insecta:
Hymenoptera)}



\author[1]{Merve Aydemir}%
\author[2]{Habeş Bilal AYDEMİR}%
\author[3]{Ertan  Korkmaz}%
\affil[1]{Gaziosmanpasa University}%
\affil[2]{Tokat Gaziosmanpaşa University}%
\affil[3]{Sivas Cumhuriyet University Faculty of Science}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
Mitochondrial transcription is being studied with the increasing
interest to better understand the coding capacity of mitochondrial DNA
(mtDNA). Based on studies showing that mtDNA encodes additional genes
that are not functional in oxidative phosphorylation (OXPHOS), it has
been understood that mitochondrial transcription is more complex than
previously thought. In this study, mitochondrial transcription was
investigated in terms of polyadenylation patterns in Apis mellifera
(Insecta: Hymenoptera). We found that both sense and antisense
transcripts were captured and polyadenylated, and gene expression levels
and polyadenylation length were highly variable between genes in Apis.
Mitochondrial RNAs of A. mellifera were found to be polyadenylated with
an average size of 31 adenines. Nevertheless, the highest transcript
representatives of each gene generally appeared to have polyadenylation
of size 5-9 bases. Generally, transcripts containing longer and/or
shorter forms of each gene were thought to indicate the existence of
putative UTR-containing genes, functional non-coding RNA genes and
variant gene forms. We showed that some transcripts contain non-template
dependent additional nucleotides before polyadenyl tail. Transcripts
containing at least some bases of the intergenic regions downstream of
the gene had the highest transcript representation after the
monocistronic gene units. These findings support the possible functional
role of the intergenic regions.%
\end{abstract}%



\sloppy


\textbf{1. INTRODUCTION}

Animal mitogenome is a highly encoded genome (\textgreater{}90\%) since
it contains no introns. It typically encodes 37 genes (22 tRNA, 13 PCG
and 2 rRNA) functional in oxidative phosphorylation (OXPHOS) process
which effects all mitochondrial function either directly or indirectly.
Thus, mitochondrial transcription is key to maintain mitochondrial
function, both in terms of the expression of oxidative phosphorylation
subunits encoded from mitochondria and mtDNA replication. Although
advances have been made with more than 40 years of work to elucidate
mitochondrial transcription, there are still challenges and
contradictions that need to be addressed (Shokolenko and Alexeyev,
2017).

In insect mitogenomes, these ``formal'' 37 genes are distributed almost
equally in heavy (GC-rich) and light (GC-poor) chains, and the entire
chains are transcribed as polycistronic units. Accordingly, almost half
of each polycistronic unit is not converted into known RNA molecules
(mRNA, tRNA or rRNA) after transcripts are processed post-
transcriptionally (Figure 1).

Interestingly, novel protein coding genes which are encoded in the
mitogenome and not functional in OXPHOS have recently been identified
(Caricasole \emph{et al.} , 2002; Lee, Yen and Cohen, 2013; Lee, Kim and
Cohen, 2016). Not only protein coding genes but also novel non-coding
RNAs have been found especially in human mitogenomes including lncRNAs
(Mercer \emph{et al.} , 2011; Rackham \emph{et al.} , 2011;
Antonicka\emph{et al.} , 2013). Even, some non-coding intergenic regions
have also been found to be present in the transcripts, especially in
mRNAs of\emph{Drosophila} (Stewart and Beckenbach, 2009). Consequently,
it has been hypothesized that the mitochondrial transcription capacity
may be more complex and intense than previously assumed.

Polyadenylation of mitochondrial transcripts can be functionally
different when compared to their nuclear counterparts. While it is known
that polyadenylation ensures mRNA stability in nuclear transcription,
mitochondrial polyadenylation can lead to the degradation of mRNAs
(Bouda, Stapon and Garcia-Diaz, 2019). Especially, non-polyadenylated
forms of some mRNAs can induce relatively higher expression and be more
stable than the polyadenylated forms, as in the case of human\emph{cox1}
(Mercer \emph{et al.} , 2011).

Even processes of the mitochondrial transcription and
post-transcriptionally RNA maturation have been studied
in\emph{Drosophila} and sea-urchin, it also needs to be studied in
different invertebrates due to the variation of transcription profiles
(Cantatore \emph{et al.} , 1990; Polosa \emph{et al.} , 2007; Torres,
Dolezal, Schl\selectlanguage{ngerman}ötterer and Ottenwälder, 2009). In this study, we predicted
the polyadenylation pattern of the mitogenome of \emph{Apis
mellifera}bioinformatically by conducting high-throughput RNA sequencing
data. Additionally, we determined the transcription patterns of
mitochondrial intergenic regions.

\textbf{2. MATERIAL and METHOD}

\textbf{2. 1. Sample selection}

\emph{Apis mellifera} RNA-seq data (SRX5798518) and \emph{Apis mellifera
sahariensis} data (NC\_035883) as reference mitogenomes were downloaded
from NCBI database. The RNA-seq data was generated using the brain
tissues of \emph{Apis mellifera} infected with deformed wing virus type
A, and produced by Traniello et al. (2020) in Illumina HiSeq 4000
platform with Chromium Next GEM Single Cell 3\selectlanguage{english}' library preparation. This
RNA-seq data was 34.2G bases in length and 18.4Gb in size. Mitogenome
reference was a verified RefSeq data and 16,569 bp in length with
non-coding A+T- rich region.

\textbf{2. 2. Determination of mitochondrial transcription pattern}

PolyA (polyT in reverse direction) selection was applied to determine
the polyadenylation patterns and relative abundances of mitochondrial
monocistronic units. Firstly, reads from the RNA-seq data were reduced
to only those containing polyA sequences with a minimum five bases in
size at any end, using the manually written script in PERL (Appendix 1).
Then, selected sequences with polyadenylation were aligned to the
reference mitogenome with ``map to reference'' option and using the
bowtie2 algorithm in Geneious R9.1.5 (\emph{https://www.geneious.com})
(Kearse \emph{et al.} , 2012) and their patterns were checked manually.
Whole RNA-seq data without polyadenylation selection was also aligned to
mitogenome, and RPKM (Reads Per Kilobase of transcript per Million
mapped reads) values were calculated for each gene.

\textbf{2.3. Prediction of potential coding capacity of mitogenome}

Mitogenome data of \emph{Apis mellifera} was analyzed in ORFFinder (Open
Reading Frame Finder, NCBI) in order to predict additional gene regions
which are able to encode proteins different than known 13 OXPHOS
proteins. Search parameters were adjusted as ``invertebrate
mitochondrial genetic code'' and ``''ATG'' and ``alternative initiation
codons''. Potential protein encoding sequences were then verified using
regular BLASTP.

\textbf{3. RESULTS and DISCUSSION}

102.775 sequences were aligned to reference mitogenome from
polyadenylation selected RNA-seq data. 76.175 of those mapped to any end
of any mitochondrial gene. 33 of 37 mitochondrial genes were represented
with at least one mapped polyadenylated sequence. It has been determined
that not only protein-coding genes but also rRNA and tRNA genes are
polyadenylated in the \emph{Apis mellifera} mitochondrial transcriptome.
In general, polyadenyls of five to 124 bases long were added to
mitochondrial genes. The highest representation for all genes were
polyadenylated forms with 5-9 adenines in length (19\%). Polyadenylated
transcripts with 10-14 and 15-19 adenines were represented by 16\% and
13\%, respectively. The length of polyadenylation and the number of
representative transcripts seemed to be inversely proportional in
logarithmic scale (Figure 2, R\textsuperscript{2}= 0,9578).
Specifically, polyadenylation length and transcript counts of each gene
were given in Figure 3.

Results obtained from polyadenylation selection and related properties
were given as a table in Appendix -2.

trnQ, trnS1, trnE and trnL2 tRNA genes were not mapped to any sense or
antisense polyadenylated end-read. trnQ, trnS1 and trnE genes are
located quite close to the noncoding A+T rich region; thus, it may be
due the reduced transcript abundance that any end-read were not mapped
to these genes. On the other hand, it is remarkable that there was no
mapped read to trnL2 gene, which is located between the most expressed
genes \emph{cox1} and \emph{cox2} . \emph{cox2} gene was represented
with 60\%, \emph{cox1} gene with 25\% and rrnL rRNA gene with 9\% of the
total end- reads; while others have less than one percent representation
(Figure 4c). It is determined that the rRNA genes were more
under-expressed than expected, probably because of the sequencing
platform used with polyA selection.

While 99.19\% of the mapped reads represent sense orientated
transcripts, antisense transcripts were also found (Figure 4d). Sense
transcripts were polyadenylated longer than antisense counterparts for
each gene group (Figure 5). The presence of both sense and antisense
transcripts indicated that functional RNA molecules were generated in
insects, similar to 7S RNA and \emph{nd6} lncRNA in humans
(Mercer\emph{et al.} , 2011). Furthermore, insect mitochondrial gene
content was distributed equally in both heavy and light chains unlike
human mitogenome which codes only \emph{nd6} and a few tRNAs in light
chain. Thus, it can be speculated that the potential of producing
noncoding- functional RNAs from insect mitogenomes can be much more
likely compared to that in human mitogenomes.

rrnL, \emph{nd4} , rrnS, \emph{nd3} and trnV genes had only sense, and
trnN, trnW and trnM genes had only antisense transcripts (Figure 4b).
Antisense transcripts had 22.85 adenines and sense transcripts had 31.00
adenines on average in their polyadenyl tails. Our data showed variable
polyadenylation length for sense and antisense transcripts of each gene,
and patterns of polyadenylation were generally independent of the
expression level (Figure 3, 4e and Appendix-2).

We found that some of mitochondrial genes predominantly appear to form
longer or shorter transcripts than annotated formal gene length. 21 of
33 mitochondrial genes tended to be found in longer transcripts. Nine of
these genes add whole sequence following the intergenic region
(\emph{atp6} , \emph{cytB} , \emph{nd4} , \emph{nd5} , \emph{nd6} ,
rrnL, trnN, trnP, trnY) and other eight of 21 add at least one base
following the intergenic region (\emph{cox3} , \emph{nd1} , \emph{nd3} ,
\emph{nd4L} , trnG, trnR, trnS2, trnT). The remaining four contain, if
they have any intergenic region, add at least one base following
mitochondrial gene in the transcript (\emph{atp8} , \emph{nd2} , trnC,
trnW) (Figure 6).

Intergenic regions that are estimated as gene rearrangement residues,
seem to be conserved despite the reduced nature of mitogenome
in\emph{Apis} . Mitochondrial regulator elements of transcription can
spread into whole genome, even into gene regions (Barshad \emph{et al.}
, 2018).Thus, it was suggested that intergenic regions may be considered
as transcription control regions (Barshad \emph{et al.} , 2018; Aydemir
and Korkmaz, 2020).

Long RNA molecules were evaluated as a result of a mis-processing of
transcription termination in human mitochondria (Temperley \emph{et al.}
, 2010). Although this assumption is acceptable for protein-coding
genes, the results of the rrnL gene is remarkable: 86.25\% and 94.02\%
of all rrnL transcripts contain whole sequence and half of whole
sequence of neighboring intergenic region, respectively. These high
rates may also suggest the possibility of incorrect annotation of this
noncoding gene since RNA gene annotations were performed using PCG
boundaries and there is no accepted, standard, consensus system for
annotation (Stewart and Beckenbach, 2009). \emph{cox1} , \emph{cox2} ,
rrnS, trnG and trnK genes tend to form a transcript that contain only
annotated sequences. Seven tRNAs seem to be transcribed mostly in
truncated forms (trnA, trnD, trnH, trnI, trnL1, trnM, trnV). Truncated
mRNA molecules are determined in human mitochondria, potentially
produced by secondary structures that may occur within the genes and
evaluated as residues of RNA surveillance pathway based on their low
frequencies with 1/161 (Szczesny \emph{et al.} , 2009; Mercer \emph{et
al.} , 2011). These polyadenylated truncated transcripts have been
evaluated as a clue for the occurrence of polyadenylation-dependent RNA
degradation processes in human mitochondria (Slomovic \emph{et al.} ,
2005). Nevertheless, it is determined that 9,80\% of all mitochondrial
transcripts of \emph{Apis} represent truncated forms. In recent studies,
it is supposed that truncated mitochondrial RNA molecules may be
precursors for circRNA molecules and are not translated (Mance \emph{et
al.} , 2020). Unlike in humans, truncated forms were found not only in
mRNAs but also in rRNA and tRNA transcripts. Truncated mitochondrial
tRNA molecules were detected and evaluated as t-elements in a freshwater
alga \emph{Cyanophora paradoxa} (Salinas-Gieg\selectlanguage{ngerman}é, Ubrig and Drouard,
2021)\emph{.} It has been proposed that these truncated tRNA molecules
potentially hybridize with neighboring mRNAs and thereby protect and
stabilize mRNAs from exonuclease digestion (Salinas-Giegé, Ubrig and
Drouard, 2021)\emph{.}

Accordingly, 75.93\% of mitochondrial transcripts containing polyadenyl
residues at their ends consist of only the annotated gene (gene +
polyA/T), 11.42\% of them consist of the gene and the entire nucleotides
of following intergenic region (gene + IG + polyA/T), 9.78\% consist of
truncated genes (gen- X base + polyA/T), 2.40\% consist of the gene and
a part of the following intergenic region (gene + X base IG + polyA/T)
and 0.47\% consist of gene, and if any intergenic region, and a part of
the neighbor gene (gene(+ IG)+ gene+ polyA/T) (Figure 4f).

Genes which transcribed in the same polycistronic unit did not show
similar expression abundance in RPKM analysis. \emph{cox2} was the most
expressed, while trnE is the least expressed mitochondrial gene, and
general pattern in expression levels was\emph{cox2}
\textgreater{}\emph{cox1} \textgreater{}\textgreater{}
trnD\textgreater{} rrnL\textgreater{} trnL2\textgreater{}
trnC\textgreater{} trnY\textgreater{} trnL1\textgreater{}
trnW\textgreater{} \emph{nd6} \textgreater{} rrnS\textgreater{}
trnI\textgreater{} \emph{nd2} \textgreater{}
trnV\textgreater{}\emph{atp6} \textgreater{} \emph{nd1} \textgreater{}
trnR\textgreater{}\emph{cytB} \textgreater{} trnN\textgreater{}
trnF\textgreater{} trnK\textgreater{} trnG\textgreater{}
trnS2\textgreater{} trnM\textgreater{} \emph{cox3} \textgreater{}
\emph{nd3} \textgreater{} trnS1\textgreater{} trnH\textgreater{}
trnP\textgreater{}\emph{nd5} \textgreater{} trnA\textgreater{}
\emph{atp8} \textgreater{}\emph{nd4} \textgreater{} trnQ\textgreater{}
trnT\textgreater{}\emph{nd4L} \textgreater{} trnE. RPKM value of each
gene was given in Table 1 and Figure 4a. Genes with high RPKM values
were found to exhibit variation in their polyadenylation profiles
(Figure 2 and Appendix 2). While rRNA expression levels are expected to
be the highest, the overexpression of \emph{cox2} and \emph{cox1} is
remarkable (Gelfand and Attardi, 1981). \emph{cox} genes, especially
\emph{cox2} mRNA accumulation can be the result of DWV (deformed wing
virus) infection of\emph{Apis} in this study. It was found that the
expression of\emph{cox2} increases due to the modulation of
prostaglandin E2 (PGE\textsubscript{2}) synthesis during viral infection
(Steer and Corbett, 2003).

According to the results of ORF Finder analyses, 205 potential ORFs were
found to have equal or longer than 75 nucleotides (25 amino acids)
(Appendix- 3). 32 of these were verified with BLAST searches, and 13 of
those were known OXPHOS genes. 13 and one of them were aligned with
bacterial and insect hypothetic proteins, respectively, and need further
validation to understand whether they are actually translated into
proteins. Four and one of 32 seemed to be variant forms of
\emph{cox1}and \emph{atp8} genes, respectively.

\textbf{3. 1. Polyadenylation patterns of gene groups}

In particular, the following findings were obtained when each
mitochondrial gene cluster was examined individually.

Polyadenyl length varied from 15.41 (\emph{atp8} ) to 41.88 (\emph{nd6}
), and 23.83 adenines for all protein-coding genes were found in sense
transcripts. Antisense transcripts of protein- coding genes had minimum
of 12 (\emph{atp6} ), mean of 20.27 and maximum of 56 (\emph{cox1} )
bases polyadenylated (Figure 3, 4e). 11 of 13 protein coding genes
seemed to be transcribed in longer units than monocistrons which include
only ORF. The excessive presence of intergenic regions in both sense and
antisense transcripts indicates that intergenic regions are also
transcribed and therefore may be functional in transcription or
translation, like recommended as a transcription termination signal in
Aydemir et al. 2022 and Roberti et al. 2003. We hypothesized that these
extra-added sequences can act as UTRs for mitochondrial mRNAs. Both 5`
and 3` UTRs are known to play critical roles in the post-transcriptional
regulation of gene expression by modulating the transport of mRNAs. It
is known that 3` UTRs regulate translation efficiency and are important
in the intracellular localization and the stability of mRNAs (Van Der
Velden and Thomas, 1999; Bashirullah, Cooperstock and Lipshitz, 2001;
Jansen, 2001). Although it is assumed that mitochondrial RNAs do not
contain UTRs, some genes may contain 3` or 5` UTR sequences (Gagliardi
\emph{et al.} , 2004; Nagaike \emph{et al.} , 2005). In general, it has
been suggested that these UTR-containing sequences are the products of
non-canonical processes and may be associated with RNA secondary
structure clusters (Mercer \emph{et al.} , 2011). 88.03\% of antisense
transcripts of \emph{Apis} have additional genomic sequences in
monocistron, and these can be considered as UTRs for insects, such as in
humans (Temperley \emph{et al.} , 2010). tRNA sequences can act as 3`
UTR through the tRNA punctuation model in human mitogenomes
(Temperley\emph{et al.} , 2010). Intergenic regions surrounding almost
all mRNAs including tRNAs may also act as UTRs for mRNAs in animal
mitogenomes, unlike that in human. It has been suggested that 3` UTR
sequences in sense transcripts may be regulated by post-transcriptional
processes and control the excessive expression of mRNAs in
\emph{Drosophila} and human (Torres, Dolezal, Schlötterer, Ottenwälder,
\emph{et al.} , 2009; Mercer\emph{et al.} , 2011). We propose that
premature stop codons which are commonly exist in mitochondrial protein
coding genes may lead to the incorporation of additional amino acids to
the protein and may produce canonically longer transcripts, such has
been previously detected in\emph{cox2} gene of \emph{D. melanogaster}
with additional 11 amino acids (Stewart and Beckenbach, 2009).

Polyadenyl length was varied from 17,49 (rrnS) to 19,35 (rrnL) with an
average of 18.42 bases for sense rRNAs. The shortest polyadenylated gene
group was rRNA transcripts (Figure 4e). This may be the result of the
tendency to economize the mitogenome due to the proportional abundance
of rRNA transcripts.

Polyadenyl length was varied from 11.64 (trnA) to 85 (trnF), and 45.08
adenines for all tRNA genes on average were found in sense transcripts.
Antisense transcripts of tRNAs have minimum of 13.25 (trnT), mean of
28.84 and maximum of 57 (trnF) bases polyadenylated (Figure 4e).
Antisense transcripts of each tRNA were much more intensely detected
than sense transcripts (10 tRNAs of 17 total). Antisense tRNA pool could
generate tRFs (tRNA- derived RNA fragments), which are a group of small
RNAs detected in human and yeast mitochondria (Lee \emph{et al.} , 2009;
Mercer \emph{et al.} , 2011; Ro \emph{et al.} , 2013; Shang \emph{et
al.} , 2018). Although the biogenesis of these RNA molecules are not
known, it was predicted that they play a role in the regulation of mtDNA
gene expression (Ro \emph{et al.} , 2013). Since tRNAs are known to be
extensively transported from the nuclear genome into the mitochondria,
the scarcity of sense tRNA molecules does not have the adverse effect on
mitochondrial transcription such as inhibition or reduction of gene
expression (Mercer \emph{et al.} , 2011).

\textbf{3.2. Non-templated additional nucleotides before
polyadenylation}

Interestingly, we found that some genes tend to add some nucleotides
just before the polyadenylation sequence. Remarkably, this bias was
determined in both sense and antisense transcripts of \emph{Apis} . We
found that 18.70\% of all mapped transcripts (14,248 sequences) contain
at least one and at most seven additional nucleotides before
polyadenyls. 42.25\% of the additional nucleotides were C, 30.28\% G,
18.28\% T and 9.19\% A nucleotide (Figure 4g). It is suggested that
additional nucleotides before polyadenyls may be functional in the
processing of 3` ends in maize mitochondrial \emph{rps12} ,
\emph{cox2}and \emph{atp9} genes (Williams, Johzuka and Mulligan, 2000).
Non-templated additional nucleotides were also found to be present in
human U2 snRNA and in some of \emph{Arabidopsis} nuclear mRNAs in other
studies (Cho \emph{et al.} , 2002; Jin and Bian, 2004). The additional
nucleotide detected in these three species mentioned above was
nucleotide C, and it was assumed that this nucleotide determines the
heterogeneity in transcript abundance (Jin and Bian, 2004). RNA-editing
mechanism is prevalent and mainly sustained with C to U editing in plant
mitochondrial transcription (Wu \emph{et al.} , 2015). While it is
assumed that animal mitochondrial transcription system shorn of
RNA-editing mechanism, this additional nucleotide sites may be templates
of post-transcriptional regulations such as RNA-editing (Lynch, Koskella
and Schaack, 2006).

\textbf{4. CONCLUSION}

In this study, the entire mitotranscriptomics of \emph{Apis
mellifera}were characterised for the first time. The following important
findings were also obtained in the present study: (i) mitochondrial
genes had polyadenyl sequences of 5 to 124 bases long, but the highest
representation for all genes was 5-9 base polyadenylated forms, (ii)
antisense transcripts were found as polyadenylated forms in transcript
pool, (iii) polyadenylation of annotated, monocistronic gene units was
the most common pattern, (iv) some of mitochondrial genes predominantly
appeared to form longer or shorter transcripts than annotated formal
gene length, (v) the overexpression of \emph{cox2} gene was potentially
due to the viral infection of selected sample, (vi)
\emph{Apis}mitogenome had the coding capacity of 32 alternative ORFs,
(vii) 18.70\% of studied transcripts contain at least one and at most
seven additional nucleotides before polyadenyls, especially the
cytosine. Apart from, comparisons from different tissues sequenced with
different approaches may provide further testing of mitochondrial gene
expression profiles due to the nature of transcriptomic analyzes.

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\textbf{LEGENDS}

Figure 1: Mitochondrial RNA pool with sense and antisense units

Figure 2: Preferred polyadenylation length among whole mitochondrial
transcripts

Figure 3: Transcript frequencies of mitochondrial genes with different
polyadenylation length

Figure 4: The summary of mitotranscriptomics of \emph{Apis mellifera}

Figure 5: Polyadenylation length of sense and antisense transcripts

Figure 6: Relatively amounts of preferred transcript patterns of
mitochondrial genes

\textbf{Data Accessibilty Statement}

Not applicable.

\textbf{Competing Interests Statement}

Not applicable

\textbf{Author Contributions Section}

Data was collected and analysed by H. Bilal Aydemir. Project
development, data management and manuscript writing was conducted by
Merve N. Aydemir. The manuscript was reviewed by E. Mahir Korkmaz.

All authors have read and approved the manuscript. A part of this
project was presented as a PhD thesis of the first author (M.N.A.).

\textbf{Acknowledgements}

We thank Dr. Mahir Budak for his valuable help in every stages of the
study. This study was financially supported by Sivas Cumhuriyet
University (CÜBAP F-573).\selectlanguage{english}
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