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 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, R2= 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 cox1 and cox2 . cox2 gene was represented with
60%, 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 nd6 lncRNA in humans (Merceret al. , 2011). Furthermore, insect mitochondrial gene content was
distributed equally in both heavy and light chains unlike human
mitogenome which codes only 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, nd4 , rrnS, 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
(atp6 , cytB , nd4 , nd5 , nd6 , rrnL,
trnN, trnP, trnY) and other eight of 21 add at least one base following
the intergenic region (cox3 , nd1 , nd3 , 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 (atp8 , nd2 , trnC, trnW) (Figure 6).
Intergenic regions that are estimated as gene rearrangement residues,
seem to be conserved despite the reduced nature of mitogenome inApis . Mitochondrial regulator elements of transcription can
spread into whole genome, even into gene regions (Barshad et al. ,
2018).Thus, it was suggested that intergenic regions may be considered
as transcription control regions (Barshad 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 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). cox1 , 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 et al. , 2009; Mercer 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 et al. ,
2005). Nevertheless, it is determined that 9,80% of all mitochondrial
transcripts of 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 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 Cyanophora paradoxa (Salinas-Giegé, Ubrig and Drouard,
2021). 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).
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. cox2 was the most
expressed, while trnE is the least expressed mitochondrial gene, and
general pattern in expression levels wascox2 >cox1 >>
trnD> rrnL> trnL2>
trnC> trnY> trnL1>
trnW> nd6 > rrnS>
trnI> nd2 > trnV>atp6 > nd1 > trnR>cytB > trnN> trnF>
trnK> trnG> trnS2>
trnM> cox3 > nd3 >
trnS1> trnH> trnP>nd5 > trnA> atp8 >nd4 > trnQ> trnT>nd4L > 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 cox2 and cox1 is remarkable (Gelfand and
Attardi, 1981). cox genes, especially cox2 mRNA
accumulation can be the result of DWV (deformed wing virus) infection ofApis in this study. It was found that the expression ofcox2 increases due to the modulation of prostaglandin E2
(PGE2) 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 cox1and atp8 genes, respectively.