LRP130, a Pentatricopeptide Motif Protein with a Noncanonical RNA-Binding Domain, Is Bound In Vivo to Mitochondrial and Nuclear RNAs

Abstract

LRP130 (also known as LRPPRC) is an RNA-binding protein that is a constituent of postsplicing nuclear RNP complexes associated with mature mRNA. It belongs to a growing family of pentatricopeptide repeat (PPR) motif-containing proteins, several of which have been implicated in organellar RNA metabolism. We show here that only a fraction of LRP130 proteins are in nuclei and are directly bound in vivo to at least some of the same RNA molecules as the nucleocytoplasmic shuttle protein hnRNP A1. The majority of LRP130 proteins are located within mitochondria, where they are directly bound to polyadenylated RNAs in vivo. In vitro, LRP130 binds preferentially to polypyrimidines. This RNA-binding activity maps to a domain in its C-terminal region that does not contain any previously described RNA-binding motifs and that contains only 2 of the 11 predicted PPR motifs. Therefore, LRP130 is a novel type of RNA-binding protein that associates with both nuclear and mitochondrial mRNAs and as such is a potential candidate for coordinating nuclear and mitochondrial gene expression. These findings provide the first identification of a mammalian protein directly bound to mitochondrial RNA in vivo and provide a possible molecular explanation for the recently described association of mutations in LRP130 with cytochrome c oxidase deficiency in humans.Transcription of protein-coding mRNAs in eukaryotic cells takes place in two distinct subcellular compartments, nuclei and mitochondria; in those cells that have them, it takes place also in chloroplasts. The vast majority of cellular mRNAs are synthesized in the nucleus, whereas mitochondria contain the genetic information for the transcription of only a few mRNAs (13 in mammalian cells), which are translated within the organelle into protein subunits of the respiratory chain (6, 45). The remaining mitochondrial proteins are encoded by nucleusderived mRNAs and are imported posttranslationally into the organelle. Thus, mitochondrial proteins are products of translation from mRNAs encoded by both nuclear and mitochondrial genomes.Nucleus-encoded mRNAs are transcribed as large precursors that undergo several processing steps before their export to the cytoplasm as mature mRNAs. These steps include addition of a 7-methylguanosine cap at the 5Ј end, cleavage and polyadenylation of the 3Ј end, and removal of introns through splicing. Throughout their maturation pathway, RNAs are associated with RNA-binding proteins as ribonucleoprotein (RNP) complexes. The proteins that are stably associated with nuclear RNAs have been extensively characterized and have been shown to participate in virtually all stages of mRNA maturation (16). In most cases, their RNA-binding activity resides in one or more distinct RNA-binding domains characterized by specific amino acid sequence motifs such as the RNP motif, KH domain, and RGG box (3). Through this binding they have an impact on the processing reactions occurring on the RNAs with which they associate (10, 16).The specific set of proteins associated with ...

Putative tumor suppressor RASSF1 interactive protein and cell death inducer C19ORF5 is a DNA binding protein

“…One of the interactions was with LRPPRC (leucine-rich PPR-motif containing protein, AAA67549) [1,9], a nucleic acid binding protein [10,11] comprised of modular domains homologous to proteins involved in cytoskeletal dynamics, nucleocytosolic shuttling, and chromosome activity [9]. Here, we confirmed that C19ORF5 interacts with LRPPRC in mammalian cells.…”

“…In the present study, we demonstrated DNA binding capacity similar to its interaction partner, LRPPRC [10][11][12], in addition to the other multiple functions of the microtubule- Like microtubule-associated proteins tau and MAP2, the role of the DNA binding capacity of C19ORF5 in cellular regulation is unclear. C19ORF5 has recently been implicated in mitochondrial aggregation associated cell death and condensation of genomic DNA followed by massive DNA degradation [3,4].…”

“…This cell death related process has been described as mitochondrial aggregation and genome destruction (MAGD), and suggested to mediate tumor suppression by the taxoid drug family and RASSF1A in response to catastrophic In addition to mitochondria-associated RASSF1 iso-forms, a yeast two-hybrid interaction screen revealed that C19ORF5 potentially interacts with several other mitochondriaassociated proteins [1]. One of the interactions was with LRPPRC (leucine-rich PPR-motif containing protein, AAA67549) [1,9], a nucleic acid binding protein [10,11] comprised of modular domains homologous to proteins involved in cytoskeletal dynamics, nucleocytosolic shuttling, and chromosome activity [9]. Here, we confirmed that C19ORF5 interacts with LRPPRC in mammalian cells.…”

“…Here, we confirmed that C19ORF5 interacts with LRPPRC in mammalian cells. Since LRPPRC [10][11][12] and other microtubuleassociated proteins (MAP2 and tau) [13,14] have been reported to interact with DNA and elevation of C19ORF5 caused massive degradation of DNA associated with its MAGD activity, we investigated whether C19ORF5 might also be a DNA binding protein with DNA degradation activity. We show that the C-terminus of C19ORF5 containing the microtubule and MAGD activities binds DNA with sufficient affinity and selectivity to be of utility in affinity purification.…”

“…The authenticity of the antibody was confirmed by immunoblot of the purified GST-C19ORF5C and other GST fused fragments and immunostain of GFP-C19ORF5C transfected COS7 cells [4]. The epitope has been mapped to within D667-S767 as shown in (a gift from Dr. Pinol-Roma, Mount Sinai School of Medicine, New York) was mapped to a sequence between amino acid residues 500 and 600 of LRPPRC by immunoprecipitation of deletion constructs synthesized by in vitro transcription-translation and by immunoblots of constructs expressed in Escherichia coli [1,10,15]. …”