OVERVIEW OF MITOCHONDRIA AND RNA EDITING WITH RESPECT TO CMS

Yowan Nerthigan. G

Contents:
Brief overview of their function and structure mtDNA structure and replication: - animals - yeast - plants

inheritance of mitochondria - petite mutants of yeast

biogenesis of mitochondria by fission

RNA editing
CMS

MITOCHONDRIA essential for cell life - ATP synthesis - many metabolic intermediates

essential for cell death

- unprogrammed death: necrosis
( eg, due to loss of energy status)

- programmed cell death

(apoptosis - controlled cell destruction)

Mitochondrial structure
Two membranes
Inner membrane invaginated Numbers of mitochondria per cell vary but usually 100s/cell

Matrix contains the TCA cycle (and other) soluble enzymes Inner membrane contains metabolite transporters and the electron transport chain

Overview of aerobic respiration

Outline of Tricarboxylic Acid Cycle

3-Carbons CO2 4-Carbons
NADH NADH + CO2

One pyruvate molecule is completely oxidised to CO2 6-Carbons

FADH NADH + CO2

The NADH and FADH produced are oxidised by the respiratory electron transport chain

Four large, multi-subunit protein complexes - complex I is a NADHubiquinone reductase - complex II is succinate dehydrogenase (part of the TCA cycle) - complex III is the ubiquinone -cytochrome c reductase - complex IV is cytochrome oxidase

The respiratory electron transport chain

Mitochondria have their own DNA and Ribosomes

Mitochondria have some of their own DNA, ribosomes, and can make many of their own proteins. The DNA is circular and lies in the matrix in structures called "nucleoids". Each nucleoid may contain 4-5 copies of the mitochondrial DNA (mtDNA).

mitochondrial DNA

Mitochondria also have their own ribosomes and tRNA: 22 tRNAs rRNAs (16S and 12S)
The ribosomes can actually be visualized in some mitochondria. In these figures, they are seen in the matrix as small dark bodies. DNA can also be visualized in mitochondria. The DNA is circular and resembles that of a bacterium in its basic structure.

This micrograph shows the DNA and ribosomes in a close-up view. Note that the circular structure of the DNA is not evident. It is noted by an arrow. There are two sets of ribosomes seen, each is circled

Mitochondrial Inheritance
Yeast has been used extensively to study mitochondrial inheritance. There is a Yeast strain, called "Petite" that have structurally abnormal mitochondria that are incapable of oxidative phosphorylation. These mitochondria have lost some or all of their DNA.

Genetic crosses between petite and wt strains showed that inheritance of this trait did not segregate with any of the nuclear chromosomes.

Mitochondrial Inheritance
Mitochondrial inheritance from yeast is biparental, and both parent cells contribute to the daughter cells when the haploid cells fuse. After meiosis and mitosis, there is random distribution of mitochondria to daughter cells. If the fusion is with yeast that are petite and yeast that are not, a certain percentage of the daughter cells will be "petite".

Mitochondrial Inheritance in Yeast

Mitochondrial Inheritance This led to the suggestion that some genetic element existed in the cytoplasm and was inherited in a different manner from nuclear genes. This is called nonMendelian inheritance or cytoplasmic inheritance.
In yeast and animals, this indicated inheritance of mitochondrial genes: in plants it also includes inheritance of chloroplast genes

Mitochondrial replication

Mitochondrial replication

cell division: random distribution of mitos between daughter cells

mitochondrial replication

Mitochondria replicate much like bacterial cells. When they get too large, they undergo fission. This involves a furrowing of the inner and then the outer membrane as if someone was pinching the mitochondrion. Then the two daughter mitochondria split. Of course, the mitochondria must first replicate their DNA. An electron micrograph depicting the furrowing process is shown in these figures.

Sometimes new mitochondria are synthesized in centres that are rich in proteins and polyribosomes needed for their synthesis. The electron micrograph in the following figure shows such a centre. It appears that the cluster of mitochondria are sitting in a matrix of proteins and other materials needed for their production.

Certain mitochondrial proteins are needed before the mitochondria can divide.
This has been shown in a study by Sorgo and Yaffe, J Cell Bio. 126: 1361-1373, 1994. They showed the result of the removal of an outer membrane protein from mitochondria called MDM10. This figure shows the results. The mitochondria are able to take in components and produce membranes and matrix enzymes. However, fission is not allowed and the result is a giant mitochondrion.

giant mitochondrion

Organisation of the mitochondrial chromosome

Human mtDNA
small, double stranded circular chromosome 16,569 bp in total
no non-coding DNA

no introns
polycistronic replication which is initiated from the D (displacement)- loop region followed by splicing of transcript to form messages.

yeast mtDNA

Yeast mitochondrial chromosome

human mtDNA

Human DNA
16,569 bp; no non-coding DNA no introns polycistronic replication followed by splicing to form messages.

Yeast mtDNA
68-75 kb, similar in structure to bacterial genome

contains introns and non-regions between genes.

Same proteins made as in animals genes transcribed separately

Despite having their own genome, most mitochondrial proteins are encoded in the nucleus, made in the cytosol and imported into the mitochondria

Synthesis of mitochondrial proteins

In all organisms, only a few of the proteins of the mitochondrion are encoded by mtDNA, but the precise number varies between organisms Subunits 1, 2, and 3 of cytochrome oxidase Subunits 6, 8, 9 of the Fo ATPase Apocytochrome b subunit of complexIII Seven NADH-CoQ reductase subunits (except in yeast) The nucleus encodes the remaining proteins which are made in the cytosol and imported into the mitochondrion. Most of the lipid is imported.

Plant mtDNA
chromosome size is much bigger but varies dramatically between species (200-2000 kb) arranged as different size circles, sometimes with plasmids. The plant mtDNA contains chloroplast sequences, indicating exchange of genetic information between organelles in plants. Much of the plant mtDNA is non-coding, but coding regions are larger than animals and fungi. Number of proteins synthesised not known definitely but more than in animals and yeast (probably about 50)

Plant mitochondria have specialised functions

in leaves they participate in photorespiration sites of vitamin synthesis (vit C, folic acid, biotin)

maize mitochondrial genome

In plants, respiration and photosynthesis operate simultaneously in the light

NIGHT

DAY

Chloroplasts are the site of photosynthesis and belong to the plastid family of organelles - they develop from proplastids in the light

thylakoid stacks

proplastid

amyloplast (in storage organs)

Rice mitochondrial and chloroplast genomes Plant mitochondria contain chloroplast genes - suggesting that genetic transfer occurs between the two organelles

Ribosom e

RuBisCO

Stroma

Chlo roplas t
Pho tos ystem II Cyto chrome b 6/ f Pho tos ystem I A syn t has e TP

Lumen

Rib osom e

Matrix

Mitochond ria
Complex I Complex I I Complex II I Complex I V Complex V

Inte rmemb rane Spa ce

Mitochondrial DNA of animals and fungi uses a different genetic code than the universal code

RNA processing in mitochondria

Plant mitochondria edit their RNA transcripts. This was first noticed when comparing cDNA sequences with genomic DNA sequences. The most common change is to replace C with U, although in some instances other changes can occur. Matrix enzymes are thought to be responsible for this, but the reason for the editing is not known. Most of the DNA in plant mitochondria is non-coding, only some of which is transcribed. RNA editing occurs even in non-coding regions such as introns.

This process was first reported in angiosperms mitochondria in 1989 The comparison between DNA and mRNA sequences of several mitochondrial genes showed some base changes in transcripts relative to the corresponding gene

The complete sequence of the moss Marchantia polymorpha revealed that no editing process ocurred in this briophyte (Oda et al., 1992). Thus, the editing process was presumed to be confined to higher plants excluding mosses. However, recent results showed that RNA editing is also operating in chloroplasts and mitochondria of bryophytes and interestingly, an important number of U to C modifications were observed (Malek et al., 1996)

Most of the editing sites reported corresponded to amino acids differing from those deduced from the gene as well as to the creation of initiation or stop codons

silent editing events not leading to a change of the encoded amino acid have been described in several plant mitochondrial mRNAs. The reason for the prevalence of silent editings is still obscure

RNA editing involving a C-to-U change may take place via different biochemical mechanisms: (i) RNA cleavage followed by cytidine release, uridine incorporation and RNA ligation, (ii) a cytidine deamination, (iii) a transamination or (iv) a transglycosilation.

Type

Compartment/ Organism

Mechanism and possible cofactors

I. INSERTION/DELETION U insertion/deletion Kinetoplastids (Trypanosoma, Leishmania, Crithidia) Mitochondria. P. Polycephalum Unknown mRNAs, rRNAs and tRNAs Paramyxoviruses Ebola viruses Mitochondria. vertebrates Cleavage, ligation guide RNAs.... Viral polymerase. Slippery transcription

C insertion (rarely U, AA CU, GU, GC insertions) G insertion

A insertion 3 mRNA poly A synthesis action

Viral polymerase. Slippery transcription Cleavage/TATase

II. BASE MODIFICATION OR REPLACEMENT C-to-U Land plants, Mitochondria,mRNAs, tRNAS, rRNAs
Chloroplasts, land plants, mRNAS

C-deamination

C-to-U

Unknown

C-to-U

Physarum Polycephalum

Unknown

C-to-U

Mammals, mRNAs (Apo-B )

Mooring sequence, Cytidine deaminase (APOBEC-1) Unknown

C-to-U

Mammals and Marsupials

U to C

Land plants. (Mitochondria, Chloroplasts), mRNA Vertebrates, mRNAs(Glutamic and serotonin receptor subunits) Hepatitis Delta Virus Drosophila, 4f-rnp

Transamination (?)

A to I

DRADA and Adeamination

A to I A to G

A-deamination of the antigenome A to I deamination (?)

U to A

Humans, alphagalactosidase Unknown mRNA (Pheto Tyr)

DRADA and other Adeaminases

A to I, A to G U to G, U Vertebrate mRNAs (Glutamic and serotonin to A receptor subunits)

CMS has been observed in a wide variety of higher plants and is characterized by the very low level or the complete absence of pollen production. As CMS may have different causes it may be assumed that it is the consequence of a mitochondrial dysfunction. EX :CMS-T of maize. In this case mitochondrial dysfunction and male-sterility are associated with mitochondrial DNA rearrangements creating a new ORF formed by fragments of rRNA genes. This ORF (T-urf13) is transcribed and translated into a chimeric polypeptide

Mitochondrial genomes encoding chimeric proteins are presumably present in all tissues of the plant. It can be speculated that the CMS phenotype affects essentially the pollen producing organs because of the high requirement of energy by this tissue. Thus, a mitochondrial dysfunction produced by a chimeric protein interfering with the organelle function by different ways, will dramatically affect pollen production while other plant organs can overcome the consequences of mitochondrial dysfunction

Assuming that the unedited form of atp9 mRNA when translated into a protein should give a poorly or nonfunctional ATP synthase subunit, we constructed nuclear transgenic tobacco plants which have been transformed with a plasmid vector carrying either the edited or the unedited forms of atp9 under the control of the constitutive cauliflower mosac virus (CaMV) promoter and terminator. The transgenic atp9 gene was linked to a signal peptide allowing the targeting of the nuclear encoded and cytoplasmically synthesized ATP 9 protein subunit to the mitochondrial compartment.

As seen in the figure, showing male sterile and fertile transgenic tobacco flowers, it was demonstrated that a significant amount of plants showed the CMS phenotype when the unedited atp9 was used, while all transgenic plants carrying the edited form of atp9 were fertile (Hernould et al., 1993).

The atp9 protein was shown to be localized in the mitochondrial fraction of transgenic tobacco using an antibody against a fragment of the signal peptide which remains linked to atp9 after the cleavage step necessary to enter into the mitochondria. The peptide tag was originated from a yeast gene which is absent in plant mitochondria and obviously in the native endogenous atp9 subunit.

Electron microscopy showed that mitochondria in the tapetum were severely affected in transgenic tobacco carrying the nuclear unedited genes, while other tissues showed the conventional mitochondrial morphology No mitochondrial modification was observed in transgenic plants carrying the edited mRNA

Respiration measurements of mitochondria isolated from transgenic plants expressing the unedited form of atp9 gave significantly lower values than control mitochondria or those from plants expressing the edited atp9 (Hernould et al., 1998).

The general characteristic of CMS is the dysfunction of mitochondria in tapetal cells. no clear relationship between the in vivo observed spontaneous CMS and a lower level of RNA editing can be established (Kurek et al., 1997). it has been reported that complete editing of an atp6 gene may restore the fertility of CMS rice (Iwabuchi et al., 1993). The production of chimeric protein, extensive recombination without creation of new ORFs, mitochondrial DNA deletions and eventually a decrease or lack of RNA editing may be some of the multiple causes of the CMS phenotype by lowering the capacity of the mitochondria to furnish energy to the cell.