Processing Of RNA Precursors Research Articles

L(1)10Bb serves as a conservative determinant for soma-germline communications via cellular non-autonomous effects within the testicular stem cell niche

The testicular stem cell niche is the central regulator of spermatogenesis in Drosophila melanogaster. However, the underlying regulatory mechanisms are unclear. This study demonstrated the crucial role of lethal (1) 10Bb [l(1)10Bb] in regulating the testicular stem cell niche. Dysfunction of l(1)10Bb in early-stage cyst cells led to male fertility disorders and compromised cyst stem cell maintenance. Moreover, the dysfunction of l(1)10Bb in early-stage cyst cells exerted non-autonomous effects on germline stem cell differentiation, independently of hub signals. Notably, our study highlights the rescue of testicular defects through ectopic expression of L(1)10Bb and the human homologous protein BUD31 homolog (BUD31). In addition, l(1)10Bb dysfunction in early-stage cyst cells downregulated the expression of spliceosome subunits in the Sm and the precursor RNA processing complexes. Collectively, our findings established l(1)10Bb as a pivotal factor in the modulation of Drosophila soma-germline communications within the testicular stem cell niche.

Splicing factor PRP-19 regulates mitochondrial stress response

Abstract Animals respond to mitochondrial perturbation by activating the mitochondrial unfolded protein response (UPRmt) to induce the transcription of mitochondrial stress response genes. In Caenorhabditis elegans, activation of UPRmt allows the animals to maintain organismal homeostasis, activate the innate immune response, and promote lifespan extension. Here, we show that splicing factors such as Precursor RNA processing 19 (PRP-19) are required for the induction of UPRmt in C. elegans. PRP-19 also modulates mitochondrial perturbation-induced innate immune response and lifespan extension. Knockdown of PRP-19 in mammalian cells suppresses UPRmt activation and disrupts the mitochondrial network. These findings reveal an evolutionarily conserved mechanism that maintains mitochondrial homeostasis and controls innate immunity and lifespan through splicing factors.

A U-Box Type E3 Ubiquitin Ligase Prp19-Like Protein Negatively Regulates Lipid Accumulation and Cell Size in Chlamydomonas reinhardtii.

Microalgae lipid triacylglycerol is considered as a promising feedstock for national production of biofuels. A hotspot issue in the biodiesel study is to increase TAG content and productivity of microalgae. Precursor RNA processing protein (Prp19), which is the core component of eukaryotic RNA splice NTC (nineteen associated complex), plays important roles in the mRNA maturation process in eukaryotic cells, has a variety of functions in cell development, and is even directly involved in the biosynthesis of oil bodies in mouse. Nevertheless, its function in Chlamydomonas reinhardtii remains unknown. Here, transcriptional level of CrPrp19 under nutrition deprivation was analyzed, and both its RNA interference and overexpressed transformants were constructed. The expression level of CrPrp19 was suppressed by nitrogen or sulfur deficiency. Cell densities of CrPrp19 RNAi lines decreased, and their neutral lipid contents increased 1.33 and 1.34 times over those of controls. The cells of CrPrp19 RNAi lines were larger and more resistant to sodium acetate than control. Considerably none of the alterations in growth or neutral lipid contents was found in the CrPrp19 overexpression transformants than wild type. Fatty acids were also significantly increased in CrPrp19 RNAi transformants. Subcellular localization and yeast two-hybrid analysis showed that CrPrp19 was a nuclear protein, which might be involved in cell cycle regulation. In conclusion, CrPrp19 protein was necessary for negatively regulating lipid enrichment and cell size, but not stimulatory for lipid storage.

Regulation of miRNA content. Part 2. Degradation of miRNAs

The scientific review presents the process of regulation of microRNA content — microRNA degradation. To write the article, information was searched using databases Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka. The article presents the characteristics of the most important process of RNA metabolism — degradation of 3'→5' RNA. Degradation of microRNA is inherent in organisms of all kingdoms of life and is involved in the regulation of RNA representation, elimination of dysfunctional or incorrectly constructed RNA molecules and processing of RNA precursors. Exoribonucleases that affect the stability of mature forms of miRNA are presented. It is emphasized that XRN exoribonucleases degrade various RNA substrates during total RNA degradation and are involved in specific processes such as nonsense-mediated degradation, gene silencing, rRNA maturation, and transcription termination. It is shown that exoribonuclease XRN2 plays a crucial role in the termination of transcription during viral infection, namely it has cytoplasmic antiviral activity against hepatitis C virus. The role of RNA-degrading exosome in microRNA degradation is presented. RNA-degrading exosome is a ubiquitous complex and 3'-5'-endo- and exoribonucleases of eukaryotes, which interacts with several processing cofactors and degrades almost all classes of cytoplasmic RNA. The article reflects the function of evolutionarily conserved phosphorolytic 3'-5'-exoribonuclease — polynucleotide phosphorylase. The role of exoribonuclease 1, which is an evolutionarily conserved 3'-5'-exoribonuclease of the DEDDh family, is involved in the final processing of 5.8S rRNA, replication-dependent histone mRNA, siRNA, and miRNA. Eri1 exoribonuclease has been shown to regulate global microRNA homeostasis in lymphocytes and to participate in NK cell development and antiviral response. Thus, one of the mechanisms of regulation of miRNA content is the most important process of RNA metabolism, which is inherent in organisms of all kingdoms of life, namely the degradation of miRNAs.

YbeY is required for ribosome small subunit assembly and tRNA processing in human mitochondria.

Mitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components of the mitochondrial ribosome require various post-transcriptional processing steps. Additional protein factors are required to facilitate the biogenesis of the functional mitoribosome. We have characterized a mitochondrially-localized protein, YbeY, which interacts with the assembling mitoribosome through the small subunit. Loss of YbeY leads to a severe reduction in mitochondrial translation and a loss of cell viability, associated with less accurate mitochondrial tRNASer(AGY) processing from the primary transcript and a defect in the maturation of the mitoribosomal small subunit. Our results suggest that YbeY performs a dual, likely independent, function in mitochondria being involved in precursor RNA processing and mitoribosome biogenesis. Issue Section: Nucleic Acid Enzymes.

Screening for mitochondrial 12S rRNA C1494T mutation in 655 patients with non-syndromic hearing loss: An observational study.

Mutations in mitochondrial DNA, especially in 12S rRNA gene, are the most important causes for hearing loss. In particular, the A1555G and C1494T mutations have been found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. To determine the frequency of C1494T mutation in deaf patients, in the current study, we screened this mutation in 655 patients with non-syndromic hearing loss and 300 control subjects. After PCR amplification of mitochondrial 12S rRNA gene and direct sequence analysis, we found that there were 2 patients carrying the C1494T mutation; however, this mutation was not detected in 300 healthy subjects. Further genetic counseling suggested that only 1 patient had an obvious family history of hearing impairment. Clinical evaluation showed that 3 of 10 matrilineal relatives suffered from hearing loss, with different age at onset of hearing loss. Molecular analysis revealed the presence of homoplasmic 12S rRNA C1494T and ND5 T12338C mutations, together with a set of polymorphisms belonging to human mitochondrial haplogroup F2. Interestingly, T12338C mutation resulted in the replacement of the first amino acid, a translation-initiating methionine with a threonine, shortening 2 amino acids of ND5 polypeptide. Moreover, this mutation is located in 2 nucleotides adjacent to the 3’ end of the mt-tRNALeu(CUN) gene. Therefore, this mutation may alter ND5 mRNA metabolism and the processing of RNA precursors. Thus, the combination of T12338C and C1494T mutations may contribute to deafness expression in this family. Taken together, our data suggested that the C1494T mutation was the molecular basis for hearing loss, screening for the mitochondrial DNA pathogenic mutations was recommended for early detection, prevention, and diagnosis of mitochondrial deafness.

Precursor RNA processing 3 is required for male fertility, and germline stem cell self-renewal and differentiation via regulating spliceosome function in Drosophila testes

The nuclear pre-mRNA spliceosome is a large complex containing five small nuclear ribonucleoprotein particles (snRNPs) and many splicing factors. Messenger RNAs (mRNAs) are generated from pre-mRNAs by the process of RNA splicing, which is conserved in eukaryotes. Precursor RNA processing 3 (Prp3) is a U4/U6-associated snRNP whose function remains largely unknown. In the present study, using genetic manipulation of a Drosophila melanogaster testis model, we demonstrated that Prp3 is essential for male fertility in Drosophila. Prp3 deficiency in germline stem cells (GSCs) and early cyst cells resulted in abnormal structure of testes and maintenance defects of GSCs and cyst stem cells. Knockdown of Prp3 in spermatogonia and early cyst cells mediated tumor formation caused by differentiation defects. Using an in vitro assay, knockdown of Prp3 decreased proliferation and increased cell death, and controlled the spliceosome function via regulating spliceosome subunits expression in Drosophila S2 cells. We also identified two other splicing factors in the Prp complex (Prp19 and Prp8), which mimicked the phenotype of Prp3 in the Drosophila stem cell niche. Our results revealed a significant role of precursor RNA processing factors in male testes, indicating that Prp3, a key spliceosome component in the Prp complex, is essential for male fertility, and germline stem cell self-renewal and differentiation, via regulating the spliceosome function in Drosophila testes.

TaPUB1, a Putative E3 Ligase Gene from Wheat, Enhances Salt Stress Tolerance in Transgenic Nicotiana benthamiana.

High salinity is one of the most severe environmental stresses and limits the growth and yield of diverse crop plants. We isolated a gene named TaPUB1 from wheat (Triticum aestivum L. cv HF9703) that encodes a novel protein containing a U-box domain, the precursor RNA processing 19p (Prp19) superfamily and WD-40 repeats. Real-time reverse transcription-PCR analysis showed that TaPUB1 transcript accumulation was up-regulated by high salinity, drought and phytohormones, suggesting that it plays a role in the abiotic-related defense response. We overexpressed TaPUB1 in Nicotiana benthamiana to evaluate the function of TaPUB1 in the regulation of the salt stress response. Transgenic N. benthamiana plants (OE) with constitutively overexpressed TaPUB1 under the control of the Cauliflower mosaic virus 35S (CaMV 35S) promoter exhibited a higher germination rate, less growth inhibition, less Chl loss and higher photosynthetic capacity than wild-type (WT) plants under salt stress conditions. These results demonstrated the increased tolerance of OE plants to salt stress compared with the WT. The OE plants had lower osmotic potential (OP), reduced Na+ toxicity and less reactive oxygen species accumulation compared with the WT, which may be related to their higher level of osmolytes, lower Na+/K+ ratio and higher antioxidant enzyme activities under salt stress conditions. Consistent with these results, the up-regulated expression of osmic- and antioxidant-related genes in OE plants indicated a role for TaPUB1 in plant salt tolerance.

Role of FAST Kinase Domains 3 (FASTKD3) in Post-transcriptional Regulation of Mitochondrial Gene Expression

The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria.

Role of a redox-based methylation switch in mRNA life cycle (pre- and post-transcriptional maturation) and protein turnover: implications in neurological disorders

Homeostatic synaptic scaling in response to neuronal stimulus or activation, and due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions (Turrigiano and Nelson, 2004). Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia, etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic; Cajigas et al., 2010). This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation, and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition, and behavior (Cajigas et al., 2010). Thus a regulatory switch, which controls the lifespan, maturation, and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at (1) the pre-transcription level, by regulating precursor-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and (2) the post-transcription level by modulating the regulatory functions of ribonucleoproteins and RNA binding proteins in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione anti-oxidant levels (Lertratanangkoon et al., 1997), the redox status of neurons might be the central regulatory switch for methylation-based changes in mRNA processing, protein expression, and turnover. Lastly, we also describe experimental methods and techniques which might help researchers to evaluate the suggested hypothesis.

Leber's Hereditary Optic Neuropathy Is Associated with the T12338C Mutation in Mitochondrial ND5 Gene in Six Han Chinese Families

To investigate the molecular pathogenesis of Leber's hereditary optic neuropathy (LHON) in Chinese families. Six Han Chinese families who seem to have maternally transmitted LHON were studied by clinical, genetic, and molecular evaluations. One hundred twenty-seven subjects from 6 Chinese families with a wide range of age-at-onset and severity of visual impairment. All subjects underwent clinical examination, genetic evaluation, and molecular analysis of mitochondrial DNA (mtDNA). The ophthalmologic examinations included visual acuity, visual field examination, visual evoked potentials, and fundus photography. The mtDNA analysis included the polymerase chain reaction (PCR) amplification of entire mtDNA and subsequent sequence determination. Six families exhibited low penetrance of visual impairment, with an average of 10.8%. In particular, 9 (6 males/3 females) of 86 matrilineal relatives in these families exhibited variable severity and age at onset in visual dysfunction. The average age at onset of visual loss was 20 years. Molecular analysis of mtDNA in these families identified the homoplasmic ND5T12338C mutation and distinct set of variants belonging to the Asian haplogroup F2. The T12338C mutation is only present in the maternal lineage of those pedigrees and not in 178 Chinese controls. This mutation resulted in the replacement of the first amino acid, a translation-initiating methionine with a threonine, shortening 2 amino acids of ND5 polypeptide. The T12338C mutation is also located in 2 nucleotides adjacent to the 3' end of the tRNA(Leu(CUN)). Thus, this mutation may alter ND5 mRNA metabolism and the processing of RNA precursors. As a result, this mutation impairs respiratory function, leading to visual impairment. Several lines of evidence suggest that the mitochondrial ND5T12338C mutation is associated with LHON. The tissue specificity of this mutation is likely due to the involvement of nuclear modifier genes. The identification of nuclear modifiers is important for the elucidation of the pathogenic mechanism of LHON and an open avenue for therapeutic interventions. The T12338C mutation should be added to the list of inherited risk factors for future molecular diagnosis. Our findings are helpful for counseling families with LHON.

Structure and Function of the Pre-mRNA Splicing Machine

Most eukaryotic pre-mRNAs contain non-coding sequences (introns) that must be removed in order to accurately place the coding sequences (exons) in the correct reading frame. This critical regulatory pre-mRNA splicing event is fundamental in development and cancer. It occurs within a mega-Dalton multicomponent machine composed of RNA and proteins, which undergoes dynamic changes in RNA-RNA, RNA-protein, and protein-protein interactions during the splicing reaction. Recent years have seen progress in functional and structural analyses of the splicing machine and its subcomponents, and this review is focused on structural aspects of the pre-mRNA splicing machine and their mechanistic implications on the splicing of multi-intronic pre-mRNAs. It brings together, in a comparative manner, structural information on spliceosomes and their intermediates in the stepwise assembly process in vitro, and on the preformed supraspliceosomes, which are isolated from living cell nuclei, with a view of portraying a consistent picture.

Nucleases of the Metallo-β-lactamase Family and Their Role in DNA and RNA Metabolism

ABSTRACTProteins of the metallo-β-lactamase family with either demonstrated or predicted nuclease activity have been identified in a number of organisms ranging from bacteria to humans and has been shown to be important constituents of cellular metabolism. Nucleases of this family are believed to utilize a zinc-dependent mechanism in catalysis and function as 5′ to 3′ exonucleases and or endonucleases in such processes as 3′ end processing of RNA precursors, DNA repair, V(D)J recombination, and telomere maintenance. Examples of metallo-β-lactamase nucleases include CPSF-73, a known component of the cleavage/polyadenylation machinery, which functions as the endonuclease in 3′ end formation of both polyadenylated and histone mRNAs, and Artemis that opens DNA hairpins during V(D)J recombination. Mutations in two metallo-β-lactamase nucleases have been implicated in human diseases: tRNase Z required for 3′ processing of tRNA precursors has been linked to the familial form of prostate cancer, whereas inactivation of Artemis causes severe combined immunodeficiency (SCID). There is also a group of as yet uncharacterized proteins of this family in bacteria and archaea that based on sequence similarity to CPSF-73 are predicted to function as nucleases in RNA metabolism. This article reviews the cellular roles of nucleases of the metallo-β-lactamase family and the recent advances in studying these proteins.

Involvement of the Mouse Prp19 Gene in Neuronal/Astroglial Cell Fate Decisions

The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19beta, a splice variant of mouse PRP19alpha corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The beta-variant possesses an additional 19 amino acid residues in-frame in the N-terminal region of the alpha-variant. The forced expression of the alpha-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the beta-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the beta-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the beta-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the alpha-variant gene plays a pivotal role in neural differentiation and that the beta-variant participates in neuronal/astroglial cell fate decisions.

Identification and Characterization of a Putative Homolog of a Spliceosome Component, Precursor RNA Processing 3 in Drosophila melanogaster

Identification and Characterization of a Putative Homolog of a Spliceosome Component, Precursor RNA Processing 3 in Drosophila melanogaster

Ribozymes: the characteristics and properties of catalytic RNAs.

Ribozymes, or catalytic RNAs, were discovered a little more than 15 years ago. They are found in the organelles of plants and lower eukaryotes, in amphibians, in prokaryotes, in bacteriophages, and in viroids and satellite viruses that infect plants. An example is also known of a ribozyme in hepatitis delta virus, a serious human pathogen. Additional ribozymes are bound to be found in the future, and it is tempting to regard the RNA component(s) of various ribonucleoprotein complexes as the catalytic engine, while the proteins serve as mere scaffolding--an unheard-of notion 15 years ago! In nature, ribozymes are involved in the processing of RNA precursors. However, all the characterized ribozymes have been converted, with some clever engineering, into RNA enzymes that can cleave or modify targeted RNAs (or even DNAs) without becoming altered themselves. While their success in vitro is unquestioned, ribozymes are increasingly used in vivo as valuable tools for studying and regulating gene expression. This review is intended as a brief introduction to the characteristics of the different identified ribozymes and their properties.

Three small nucleolar RNAs that are involved in ribosomal RNA precursor processing.

Three small nucleolar RNAs (snoRNAs), E1, E2 and E3, have been described that have unique sequences and interact directly with unique segments of pre-rRNA in vivo. In this report, injection of antisense oligodeoxynucleotides into Xenopus laevis oocytes was used to target the specific degradation of these snoRNAs. Specific disruptions of pre-rRNA processing were then observed, which were reversed by injection of the corresponding in vitro-synthesized snoRNA. Degradation of each of these three snoRNAs produced a unique rRNA maturation phenotype. E1 RNA depletion shut down 18 rRNA formation, without overaccumulation of 20S pre-rRNA. After E2 RNA degradation, production of 18S rRNA and 36S pre-rRNA stopped, and 38S pre-rRNA accumulated, without overaccumulation of 20S pre-rRNA. E3 RNA depletion induced the accumulation of 36S pre-rRNA. This suggests that each of these snoRNAs plays a different role in pre-rRNA processing and indicates that E1 and E2 RNAs are essential for 18S rRNA formation. The available data support the proposal that these snoRNAs are at least involved in pre-rRNA processing at the following pre-rRNA cleavage sites: E1 at the 5' end and E2 at the 3' end of 18S rRNA, and E3 at or near the 5' end of 5.8S rRNA.

Post-transcriptional Regulation of Human Interleukin-2 Gene Expression at Processing of Precursor Transcripts

Interleukin-2 (IL-2) regulates the clonal expansion of activated T cells and is produced in limited amounts during an immune response. Mitogenic induction of human IL-2 gene expression elicits a transient wave of unstable mRNA. We show here that transcription continues unabated during and well beyond the time when the wave is subsiding, yet few, if any, new mRNA molecules are generated once the wave has reached its maximum. Instead, IL-2 precursor transcripts accumulate, becoming the majority of expressed IL-2 RNA molecules. The flow of precursor transcripts into mature mRNA becomes inhibited in the course of induction. When translation is blocked (e.g. by cycloheximide), expression of IL-2 mRNA can be superinduced by 2 orders of magnitude. This superinduction is completely dependent upon transcription, yet is not accompanied by any significant increase in the rate of primary transcription or in mRNA stability. Instead, the processing of nuclear IL-2 precursor transcripts is greatly facilitated, resulting in pronounced superinduction of cytoplasmic mRNA. Once its transcription has been induced, therefore, expression of the IL-2 gene is down-regulated extensively at the level of precursor RNA processing.

Ribosomal RNA precursor processing by a eukaryotic U3 small nucleolar RNA-like molecule in an archaeon

An RNA-containing endonuclease that catalyzes the excision and maturation of the 16S ribosomal RNA (rRNA) from the rRNA primary transcript (pre-rRNA) in the hyperthermophilic archaeon Sulfolobus acidocaldarius has been characterized. The ribonucleoprotein was inactivated by micrococcal nuclease treatment and inactivation was reversed by reconstitution with bulk RNA. A 159-nucleotide RNA with sequence and structural similarity to U3 small nucleolar RNAs of eukaryotes copurified with the endonuclease activity. Oligonucleotide-targeted ribonuclease H inactivation of the U3-like RNA component also abolished processing activity. A motif within the U3 homolog is complementary to the region around the three cleavage sites in the pre-RNA substrate. Thus, U3-mediated processing of pre-rRNA is not specific to eukaryotes; its origin predates the divergence of archaea and eukaryotes.

Suppression of a yeast mitochondrial RNA processing defect by nuclear mutations.

The S. cerevisiae strain h56 is a temperature-sensitive mit- mutant containing a single nucleotide substitution in the region 5' to the reading frame of the mitochondrial var1 gene. The mutation decreases the efficiency of processing of a precursor RNA such that little var1 mRNA is produced at the restrictive temperature, 36 degrees C. This communication reports the isolation and characterization of several strains carrying nuclear mutations which suppress the temperature-sensitivity of h56. Both dominant and recessive suppressor mutations were isolated. One dominant suppressor strain (h56-S4) was characterized biochemically, and the mechanism of suppression shown to involve a restoration of precursor RNA processing at the restrictive temperature, with a concomitant increase in the synthesis of the var1 protein. It appears likely that the suppressing allele encodes a component of an RNA processing endoribonuclease active on var1 transcripts. A genomic library was constructed from the h56-S4 strain, and several plasmids showing suppressed activity were isolated. A preliminary analysis of these plasmids is presented.