We report here on the identification of a novel human nuclear-encoded mitochondrial poly(A) polymerase. Immunocytochemical experiments confirm that the enzyme indeed localizes to mitochondrial compartment. Inhibition of expression of the enzyme by RNA interference results in significant shortening of the poly(A) tails of the mitochondrial ND3, COX III and ATP 6/8 transcripts, suggesting that the investigated protein represents a bona fide mitochondrial poly(A) polymerase. This is in agreement with our sequencing data which show that poly(A) tails of several mitochondrial messengers are composed almost exclusively of adenosine residues. Moreover, the data presented here indicate that all analyzed mitochondrial transcripts with profoundly shortened poly(A) tails are relatively stable, which in turn argues against the direct role of long poly(A) extensions in the stabilization of human mitochondrial messengers.

The mechanism of human mitochondrial RNA turnover and surveillance is still a matter of debate. We have obtained a cellular model for studying the role of hSuv3p helicase in human mitochondria. Expression of a dominant-negative mutant of the hSUV3 gene which encodes a protein with no ATPase or helicase activity results in perturbations of mtRNA metabolism and enables to study the processing and degradation intermediates which otherwise are difficult to detect because of their short half-lives. The hSuv3p activity was found to be necessary in the regulation of stability of mature, properly formed mRNAs and for removal of the noncoding processing intermediates transcribed from both H and L-strands, including mirror RNAs which represent antisense RNAs transcribed from the opposite DNA strand. Lack of hSuv3p function also resulted in accumulation of aberrant RNA species, molecules with extended poly(A) tails and degradation intermediates truncated predominantly at their 3′-ends. Moreover, we present data indicating that hSuv3p co-purifies with PNPase; this may suggest participation of both proteins in mtRNA metabolism.

The THO complex is a key factor in co-transcriptional formation of export-competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In yeast, this complex has been described as a heterotetramer (Tho2, Hpr1, Mft1, and Thp2) that interacts with Tex1 and mRNA export factors Sub2 and Yra1 to form the TRanscription EXport (TREX) complex. In this study, we purified yeast THO and found Tex1 to be part of its core. We determined the three-dimensional structures of five-subunit THO complex by electron microscopy and located the positions of Tex1, Hpr1, and Tho2 C-terminus using various labelling techniques. In the case of Tex1, a b-propeller protein, we have generated an atomic model which docks into the corresponding part of the THO complex envelope. Furthermore, we show that THO directly interacts with nucleic acids through the unfolded C-terminal region of Tho2, whose removal reduces THO recruitment to active chromatin leading to mRNA biogenesis defects. In summary, this study describes the THO architecture, the structural basis for its chromatin targeting, and highlights the importance of unfolded regions of eukaryotic proteins.

Little is known about the RNA polymerase III (Pol III) complex assembly and its transport to the nucleus. We demonstrate that a missense cold-sensitive mutation, rpc128-1007, in the sequence encoding the C-terminal part of the second largest Pol III subunit, C128, affects the assembly and stability of the enzyme. The cellular levels and nuclear concentration of selected Pol III subunits were decreased in rpc128-1007 cells, and the association between Pol III subunits as evaluated by coimmunoprecipitation was also reduced. To identify the proteins involved in Pol III assembly, we performed a genetic screen for suppressors of the rpc128-1007 mutation and selected the Rbs1 gene, whose overexpression enhanced de novo tRNA transcription in rpc128-1007 cells, which correlated with increased stability, nuclear concentration, and interaction of Pol III subunits. The rpc128-1007 rbs1⌬ double mutant shows a synthetic growth defect, indicating that rpc128-1007 and rbs1⌬ function in parallel ways to negatively regulate Pol III assembly. Rbs1 physically interacts with a subset of Pol III subunits, AC19, AC40, and ABC27/Rpb5. Additionally, Rbs1 interacts with the Crm1 exportin and shuttles between the cytoplasm and nucleus. We postulate that Rbs1 binds to the Pol III complex or subcomplex and facilitates its translocation to the nucleus.A ll eukaryotic cells have at least three different RNA polymerases (Pol). Pol I synthesizes the large precursor of rRNA, Pol II produces mainly mRNAs, and Pol III generates tRNAs, 5S rRNA and other small noncoding RNAs.Pol III, which is the focus of this work, is a heteromultimeric protein complex that contains 17 distinct subunits in Saccharomyces cerevisiae. The two largest subunits, C160 and C128, form opposite sides of the active-center cleft and harbor the catalytic activity, and they are related to the ␤= and ␤ components of the ␣ 2 ␤␤= core of bacterial RNA polymerase. Homology to the bacterial ␣ subunit, although less strong, was also observed, with the subunit AC40 common for yeast and Pol III and Pol I. A second ␣-like subunit, AC19, forms a heterodimer with AC40, which is a functional equivalent to the ␣ 2 homodimer in prokaryotes (1). The polymerase core, in addition to the ␣ 2 ␤␤=-like structure, contains six small subunits, which are structurally and functionally conserved from yeast to humans. The small core subunits either bind or bridge the two largest catalytic subunits. In addition to the central core, the remaining subunits of Pol III are organized in heterodimeric or trimeric stalks or subdomains. Significantly, the C37 to C53 and C82 to C34 subdomains, which are stably bound to the RNA Pol III core, resemble Pol II general transcription factors TFIIF and TFIIE (2).Little is known about how polymerase complexes are assembled from the subunits, how they reach their functional destination, and how they dissociate after transcription. In bacteria, the assembly of RNA polymerase starts with the formation of the ␣␣ dimer, which then interacts with the ␤ subunit to yield an ␣␣␤...