Comparative mitochondrial zygomycetes: bacterial-like Rnase P RNAs, mobile elements and a close source of the group I intron invasion in angiosperm

1
Comparative mitochondrial zygomycetes
bacterial-like Rnase P RNAs, mobile elements and
a close source of the group I intron invasion in
angiosperm
Seif et al., (2005) NAR 33(2) 734-744

• Presented by
• Somayeh Haji Kazem Nili
• Kalyani Rajalingham

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Summary

• The mtDNAs from three distantly related
  zygomycetes have been sequenced
• Rhizopus oryzae (R.oryzae)
• Mortierella verticillata (M.verticillata)
• Smittium culisetae (S.culisetae)
• AIM
• Analyze comparative mitochondrial genome
• Remediate the lack of data for phylogenetic
  inferences
• Facilitate biochemical investigations

Seif et al., (2005) NAR 33(2) 734-744
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Background

• Fungi constitute four phylum of highly diverse
  organisms
• Ascomycetes complete nuclear and mitochondrial
  sequences of more than dozen are known
• Chytrids few mitochondrial sequences are known
• zygomycete
• No mitochondrial gene sequences are known
• Nuclear gene sequences are limited (mostly rRNA
  sequences)
• Impossible to determine if zygomycota is a
  monophyletic taxon
• Sequence complete mtDNAs sequencing from several
  zygomycetes to remediate this situation

Ascomycota Zygomycota
Basidiomycota Chatridiomycota
Seif et al., (2005) NAR 33(2) 734-744
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Research components

• Compare mitochondrial genomes
• Seven newly identified zygomycete mitochondrial
  RNase P (mtP-RNAs)
• Are zygomycetes monophyletic?
• Group I introns invasion of cox1 gene in
  angiosperm originated in zygomycete close to
  Rhizopus

Gene content Genetic code
Gene organization Conserved 3RNA processing site
Expression Secondary structure
Seif et al., (2005) NAR 33(2) 734-744
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Introduction

• Mitochondrial phylogenies
• Base on 13 protein sequences
• Resolves deep divergences in the fungal and
  animal lineage
• Zygomycete mtDNA are of considerable interest for
  comparative gene expression studies
• Zygomycete mtDNA contains rnpB gene
• Encodes the RNA subunit of RNase P
• Absent in basidiomycete and chytridiomycete
• Patchy distribution in ascomycetes
• No published data on zygomycetes
• The RNA subunit of mitochondrial RNase P
  (mtP-RNA)
• The enzymatically active part of an endonuclease
• Has various size and sequences
• Complicated identification

Seif et al., (2005) NAR 33(2) 734-744
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Zygomycete mtDNAsShape and Size

• Are linear multimeric concatemers in vivo
• Mapped as circular molecules
• Size ranges between 54-58 kb
• Coding regions
• R.oryzae 40.6
• M.verticillata 43.1
• S.culisetae 35.3
• Coding regions nad2/nad3 and nad4L/nad5 of
  R.oryzae overlap by 1 nt

Seif et al., (2005) NAR 33(2) 734-744
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Zygomycete mtDNAsComponents

• The basic fungal set of genes
• Full set of tRNAs
• RNA component of mitochondrial RNase P (rnpB)
• RNA component of ribosomal protein (rps3)
• rps3 lacking in R.oryzae

Creator/Presenter Somayeh Haji Kazem Nili
Seif et al., (2005) NAR 33(2) 734-744
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mtDNAs Genomic Maps

• Inner circle scale (kb)
• Outer circle location of genes
• Black exons
• Gray introns and intronic ORFs
• Green ORFs
• Blue rps3
• Red rnpB
• ORFs, rps3 and rnpB are colored to distinguish
  them from standard fungal genes (black).

Seif et al., (2005) NAR 33(2) 734-744
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Standard Protein Coding Genes

• R.oryzae and S.culisetae retain standard
  translation code for protein coding genes
• M.verticillata reassigned two UGA stop codons
  as tryptophan Trp on
• nad3
• nad4
• UGA (Trp) codons are also exist in S.culisetae
  intronic ORFs
• ORF283
• ORF248

Seif et al., (2005) NAR 33(2) 734-744
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UGA (Trp) of Intronic ORFs (283 248)

• Encoding group I introns homing endonucleases of
  the LAGLI-DADG type
• UGA (Trp) at amino acid position 237 of ORF248
• Distinctive motif of this class of endonuclease
• Highly conserved
• Possibly is a vestige of horizontal intron
  transfer from a fungus adapted to this
  translation code
• Closely related to ORF313 of Podospora anserina
• S.culisetae and M.verticillata do not encode
  trnW(uca)
• trnW(uca) tRNA recognizing UGA and UGG (Trp)
• Assumed UGA codons are inefficiently encoded by
  trnW(uca)
• Alternatively modified or partially edited C in
  the wobble position of the anticodon may allow
  efficient recognition of UGA (Trp)

Seif et al., (2005) NAR 33(2) 734-744
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tRNA Complete Set

• Zugomycete mtDNAs encode sufficient tRNA to
  recognize all encountered codons
• trnI(cau) is absent in R.oryzae
• ATA(Ile) codons are absent in standard
  mitochondrial genes
• ATA(Ile) occur in intronic ORFs
• The tRNA require for ATA(Ile) translation is
  imported from cytoplasm to recognize these codons
• The intronic ORFs are neither translated nor
  required for intron splicing
• These codon positions are recognized by other
  tRNAs at low efficiency, resulting amino acid
  misincorporation
• Such unexpected codon usage reflects horizontal
  intron transfer from species that are adapted to
  the use of UGA(Trp) and/or ATA(Ile)

Seif et al., (2005) NAR 33(2) 734-744
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mtP-RNAs

• Mitochondrial RNA subunit of RNase P
• Encoded by rnpB genes
• rnpB genes are from outside fungi
• Only present in the green alga and various
  jakobids
• In some ascomycetes
• Absent in basidiomycetes and chytridiomycetes
• Their presence in all three zygomycetes is
  striking

Seif et al., (2005) NAR 33(2) 734-744
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mtP-RNA

• S.culisetae
• Close to smallest nrp gene (145 nt)
• Has highly reduced structure
• Secondary structure identical to budding yeasts
  mtP-RNA
• Perfectly matching the minimum consensus
  secondary structure of fungal mtP-RNAs
• M.verticillata and R.oryzae
• Largest nrp gene (980 and 830 bp)
• Secondary structure identical to bacterial
  mtP-RNA
• 3 end of mtP-RNA in M.verticillata is 9 nt
  longer than proposed secondary structure
• Elongated by a cytidin-rich stretch sequence
• 5 end of mtP-RNA in M.verticillata is and both
  3 and 5ends of nrp gene in R.oryzae are match
  the proposed secondary structure and reveal
  little heterogeneity of mtP-RNA termini

Seif et al., (2005) NAR 33(2) 734-744
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mtP-RNA Secondary Structure

Seif et al., (2005) NAR 33(2) 734-744
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mtP-RNA Secondary Structure

• Zygomycete mtP-RNA structures cover an
  unprecedented wide range of intermediate stages
  in loss of RNA structural elements
• R.oryzae mtP-RNA structure
• The most bacteria like secondary structures
• Containing almost all structural elements of the
  bacterial minimum secondary structure consensus
• Lacks P13, P14 and P19 helixes (putative-alpha
  helical structures)
• If R.oryzae mtP-RNA structure had these sites it
  was resemble to protist mtP-RNA
• M.verticillata mtP-RNA structure
• Resemble to mtP-RNA of ascomycete (Taphrina
  deformans)
• Lacks omnipresent P2 sihelix
• S.culisetae mtP-RNA structure
• The tiny yeast like mtP-RNA molecule
• Lacks the omnipresent P2 helix
• P2 helix absence in M.verticillata and
  S.culisetae mtP-RNA structure indicates its loss
  in a common ancestor

Seif et al., (2005) NAR 33(2) 734-744
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mtP-RNA Insertion Regions

• The large size of M.verticillata and R.oryzae
  mtP-RNA is due to insertions at the J5-15 and
  J5-18 junctions in the P12 helix
• These insertion sequences can be folded into
  double hairpin structure
• mtP-RNA cDNA sequence of R.oryzae were amplified
  by PCR to determine whether these regions are
  conserved regions or more variable insertion
  elements or introns
• They are not conserved regions, as their presence
  in mtP-RNAs pinpoints structural regions are not
  critical for RNase P activity
• They are not introns because the cDNA sequence is
  identical to the genomic sequence
• They are variable insertion elements
• Variations in insertion points and sizes
  indicates that they have been acquired recently
  and independently

Seif et al., (2005) NAR 33(2) 734-744
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Conserved C-rich motifs in mRNAs and SSU-rRNA
SSU-rRNA Small subunit of Ribosomal RNA
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Conserved C-rich motifs in mRNAs and SSU-rRNA

• downstream of mt-protein- and SSU-rRNA coding
  regions
• site of 3' RNA processing, and are retained in
  the mature RNA molecules

C-rich clusters
SSU-rRNA Small subunit of Ribosomal RNA
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genes of all three species contain numerous
mobile group I introns
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Introns that move homing and retrohoming.
Certain introns include a gene (shown in red) for
enzymes that promote homing (certain group I
introns) or retrohoming (certain group II
introns). (a) The gene in the spliced intron is
bound by a ribosome and translated. Group I
homing introns specify a site-specific
endonuclease, called a homing endonuclease. Group
II retrohoming introns specify a protein with
both endonuclease and reverse transcriptase
activities.
Ref http//quizlet.com/16660163/chapter-26-slides
-flash-cards/
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Refhttp//nar.oxfordjournals.org/content/29/18/37
57/F1.large.jpg
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Ref http//home.cc.umanitoba.ca/hausnerg/
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Generalized homing mechanisms for mobile group I
introns and inteins. In both cases, the activity
of the endonuclease (which is translated either
as a free-standing protein from the intron, or as
a fusion with the surrounding intein) leads to a
double-strand break in an allele of the host gene
that does not contain the intervening sequence.
Subsequent repair via homology-driven strand
invasion and recombination and DNA replication,
using the allele containing the intron or intein
(as well as the associated endonuclease coding
sequence), completes the homing process. HEG,
homing endonuclease gene.
Ref Stoddard Mobile DNA 2014 57  
doi10.1186/1759-8753-5-7/
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ORF encodes endonuclease - for mobility
Closely Related
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C-terminus of a foreign atp6 gene
atp6 in phase with the resident gene - functional
hybrid gene
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ORF376 encodes a protein related to homing
endonucleases
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Have evidence for endonucleolytic activity
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we currently do not have biochemical evidence
for the endonucleolytic activity
29
With 14 introns, the mtDNA of S.culisetae
contains the largest number, 9 of which are
located in the cox1 gene
30
In R.oryzae, we identified one intron
cox1-i1(ORF305), which is most similar to
introns inserted at the same positions of
angiosperm cox1 genes (highest BLAST expect
value of e-114 with Philodendron oxycardium,
Lamium sp. and Malpighia glabra).
31
. Because this is the only group I intron in
vascular plant mtDNAs, it has most likely been
acquired by lateral transfer.
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R.oryzae
Phylogeny of intronic ORFs. Sequences of
intronic ORFs inserted in cox1 genes were
obtained from the following species R.oryzae,
S.culisetae, Monoblepharella15,
Schizosaccharomyces japonicus , S.octosporus ,
P.anserina, Pichia canadensis, S.cerevisiae,
Chlorella vulgaris, Prototheca wickerhamii,
Peperomia obtusifolia, Veronica catenata and
Maranta Leuconeura. Sequences were aligned as
described in Materials and Methods, and a
phylogeny was inferred by ML. This tree robustly
supports the monophyly between sequences from
Monoblepharella15, R.oryzae, and the angiosperms,
strongly suggesting horizontal intron transfer
between these two groups
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Phylogeny of intronic ORFs. Sequences of
intronic ORFs inserted in cox1 genes were
obtained from the following species R.oryzae,
S.culisetae, Monoblepharella15,
Schizosaccharomyces japonicus , S.octosporus ,
P.anserina, Pichia canadensis, S.cerevisiae,
Chlorella vulgaris, Prototheca wickerhamii,
Peperomia obtusifolia, Veronica catenata and
Maranta Leuconeura. Sequences were aligned as
described in Materials and Methods, and a
phylogeny was inferred by ML. This tree robustly
supports the monophyly between sequences from
Monoblepharella15, R.oryzae, and the angiosperms,
strongly suggesting horizontal intron transfer
between these two groups
34
ORF305 from R.oryzae is the closest and most
similar relative of the angiosperm ORFs,
suggesting that the fungal donor of the group I
intron and its resident ORF was a zygomycete
35
Phylogenetic analysis with standard mitochondrial
proteins Are zygomycetes paraphyletic?
Testing the monophyly of Zygomycota
36
R.oryzae
Mortierella verticillata
Allomyces macrogynus
Smittium culisetae
Fungal phylogeny based on multiple proteins.
Mitochondrion-encoded protein sequences from
Harpochytrium sp. 94, Crinipellis perniciosa,
Cryptococcus neoformans, Hypocrea jecorina,
Amoebidium parasiticum, Spizellomyces punctatus,
Yarrowia lipolytica, Monosiga brevicolis,
R.oryzae, Rhizophydium sp. JEL136, Penicillium
marneffei, Pichia canadensis, Cantharellus
cibarius, Sarcophyton glaucum, S.culisetae,
Monoblepharella15, Metridium senile,
A.macrogynus, Hyaloraphidium curvatum, Candida
albicans, P.anserina, S.commune and
M.verticillata were aligned, concatenated and
trimmed. Phylogenies were inferred from the
resulting 2890 character alignment using four
different methods. Shown here is the ML tree
inferred using IQPNNI, along with bootstrap
support values from PHYML, IQPNNI, MrBayes and
separate ML analysis, in order from top to
bottom, based on 100 replicates. Nodes with 100
bootstrap support using all methods are indicated
by an asterisk. Clearly, both the position of
A.macrogynus and the branching order of the
zygomycetes remain unclear, although the topology
is robust overall.
37
R.oryzae
Mortierella verticillata
Allomyces macrogynus
Smittium culisetae
two major areas of uncertainty the position of
A.macrogynus and the relative branching
positions of the zygomycetes
38
R.oryzae
Mortierella verticillata
Allomyces macrogynus
Smittium culisetae
Although S.culisetae and M.verticillata are
monophyletic in the best tree under this model
of separately optimized functional classes, all
topologies in which all three zygomycetes are
monophyletic are rejected
39
Clearly, these data are insufficient to resolve
the phylogeny of the zygomycetes, most likely
because these three species diverge deeply
within fungi, and at relatively short distance
from each other. In such a situation, two
strategies can be used to resolve the dilemma,
addition of more zygomycete and neighboring
fungal lineages or addition of more sequence per
species
40
Time to RelaxJoke
So, any questions?