Functional Analysis of the MTERF Protein Family in Cultured Human Cells

Abstract

Väitöskirjatyössä Functional analysis of the MTERF protein family in cultured human cells oli tavoitteena karakterisoida ihmisen MTERF-proteiiniperheen jäsenten tehtäviä mitokondrion DNA:n (mtDNA) metaboliassa viljellyissä ihmissoluissa molekyylibiologian keinoin.

Väitöskirjatyön ensimmäisessä osassa tutkittiin MTERF:n (mitochondrial transcription termination factor) sitoutumista mtDNA:han ja sen mahdollista roolia mitokondrion genomin replikaatiossa. Toisessa osassa valotettiin MTERF-proteiinin tehtäviä mtDNA:n transkriptiossa. Työn kolmannessa osassa tutkittiin kahden varsin äsken löydetyn MTERF-proteiiniperheen jäsenen, MTERFD1:n ja MTERFD3:n, sitoutumista mtDNA:han ja niiden tehtäviä mtDNA:n ylläpidossa. Lisäksi väitöskirjan neljännessä osassa valotettiin TFAM:n roolia mtDNA:n metaboliassa.

MTERF:n havaittiin sitoutuvan mtDNA:ssa useisiin kohtiin kanoonisen sitoutumiskohtansa lisäksi sekä in vitro että in vivo. Merkittävää oli myös havainto, että MTERF edistää mitokondriaalisen replikaation taukoamista sekä kanoonisessa että uusissa sitoutumiskohdissaan. TFAM:n voimistamat replikaation pysäytyskohdat olivat vertailtaessa varsin diffuuseja toisin kuin MTERF:n vastaavat. MTERF-proteiinitasojen muuntelu in vivo vaikutti vain lievästi mitokondriaaliseen transkriptioon. Tulokset implikoivat, että MTERF-proteiinin määrä ei säätele mitokondriaalisten transkriptien suhteellisia tasoja millään yksinkertaisella tavalla vaan että siihen liittyy kompensaatiomekanismeja. Siinä missä MTERF-proteiinin määrän muutoksella oli vain vähäinen vaikutus mitokondrion transkriptitasoihin, TFAM:n ylituotannolla oli puolestaan selkeä vaikutus.

MTERFD1:n ja MTERFD3:n havaittiin olevan mitokondriaalisesti kohdennettuja proteiineja, mutta kummallekaan näistä proteiineista ei löydetty sekvenssispesifejä sitoutumiskohtia. MTERFD3:n ja hieman vähemmissä määrin MTERFD1:n ylituotannon havaittiin vähentävän mtDNA:n kopiolukumäärää ja estävän mtDNA:n replikaation loppuunsaattamista. Tulokset implikoivat, että myös näillä uusilla MTERF-proteiiniperheen jäsenillä on rooli mtDNA:n replikaatiossa.

Human mitochondrial DNA (mtDNA) is a double-stranded circular molecule of ~16 kb. In the major coding strand of human mtDNA there are two transcription units, one of which is dedicated to the synthesis of ribosomal RNAs and two transfer RNAs (the ´rRNA transcription unit´) and the other one to the synthesis of all messenger RNAs and the remaining transfer RNAs (the ´mRNA transcription unit´). The initiation sites for these two transcription units are located near each other and the transcription units partially overlap. They are independently controlled and differentially expressed. The central aim of the present project was to study the functional roles of human mitochondrial transcription termination factor (MTERF), the protein that is believed to control the relative activities of the two transcription units in the major coding strand of mtDNA.

MTERF is a DNA-binding protein that interacts with mtDNA as a monomer. It binds to a 28 bp region within the leucine (UUR) transfer RNA (tRNALeu(UUR)) gene at the position immediately adjacent and downstream of the 16S ribosomal gene. In vitro MTERF has been shown to promote transcription termination but so far no evidence has been reported supporting the idea that it performs such a role in vivo. The A3243G MELAS (mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes) mutation is located within the MTERF binding sequence in mtDNA. It has been suggested that elucidating the physiological function(s) of MTERF could help to understand the pathogenesis of MELAS syndrome. It has been shown that the A3243G mutation reduces the binding affinity of MTERF to its target sequence, which should mean that the efficiency of rRNA transcription termination decreases.

MTERF belongs to a family of related proteins whose physiological functions are unclear. This study addressed the issue of the functional role of MTERF and that of two novel MTERF protein family members MTERFD1 and MTERFD3 in vivo at the cellular level. The effect of MTERF over-expression and knock down in HEK293T-derived cells was studied on steady-state mitochondrial transcript levels and after mtDNA and RNA depletion with EtBr. Modulating MTERF levels in vivo had a modest effect on mitochondrial transcription. It may be inferred that MTERF levels do not determine the relative levels of transcripts representing the two different transcription units of the heavy strand in a simple manner but that compensatory mechanisms are involved. Whereas altering MTERF levels had only minor effects on mitochondrial transcript levels, over-expression of TFAM had a clear effect by slowing down the recovery of the tRNA levels after EtBr-induced depletion of mitochondrial DNA and RNA.

Using two-dimensional neutral agarose gel electrophoresis (2DNAGE), MTERF over-expression or knockdown was found to affect mtDNA replication pausing, although no effect on mtDNA copynumber was detected. MTERF was inferred to promote pausing both at the canonical MTERF-binding site as well as at novel, weaker binding sites identified by electrophoretic mobility shift assay (EMSA) and by using systematic evolution of ligands by exponential enrichment (SELEX). In contrast to MTERF over-expression enhanced replication pause sites, the pause sites enhanced by TFAM over-expression were found comparatively diffuse.

Immunocytochemistry showed that epitope-tagged MTERFD1 and MTERFD3 are mitochondrially targeted, but EMSA and SELEX did not identify plausible sites of sequence-specific DNA binding for either of these proteins. Over-expression of epitope-tagged MTERFD3 or, to a lesser extent, MTERFD1 in HEK293T-derived cells was found to decrease mtDNA copynumber and to impair the completion of mtDNA replication, based on the accumulation of specific classes of replication intermediates, as revealed by 2DNAGE.

In conclusion, the results presented in this thesis further elucidate the role of MTERF in mitochondrial transcription and moreover establish that MTERF has a role also in mtDNA replication. These findings are further analyzed in light of TFAM results. A solid ground for further studies on MTERFD1 and MTERFD3 is laid here as results reported in this thesis indicate that MTERFD1 and MTERFD3 have a role in mtDNA replication too.