Nuclear RNA Surveillance in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e: Trf4p-dependent Polyadenylation of Nascent Hypomethylated tRNA and an Aberrant Form of 5S rRNA

1-Methyladenosine modification at position 58 of tRNA is catalyzed by a two-subunit methyltransferase composed of Trm6p and Trm61p in Saccharomyces cerevisiae. Initiator tRNA (tRNAiMet) lacking m1A58 (hypomethylated) is rendered unstable through the cooperative function of the poly(A) polymerases, Trf4p/Trf5p, and the nuclear exosome. We provide evidence that a catalytically active Trf4p poly(A) polymerase is required for polyadenylation of hypomethylated tRNAiMet in vivo. DNA sequence analysis of tRNAiMet cDNAs and Northern hybridizations of poly(A)+ RNA provide evidence that nascent pre-tRNAiMet transcripts are targeted for polyadenylation and degradation. We determined that a mutant U6 snRNA and an aberrant form of 5S rRNA are stabilized in the absence of Trf4p, supporting that Trf4p facilitated RNA surveillance is a global process that stretches beyond hypomethylated tRNAiMet. We conclude that an array of RNA polymerase III transcripts are targeted for Trf4p/ Trf5p-dependent polyadenylation and turnover to eliminate mutant and variant forms of normally stable RNAs

Nuclear Surveillance and Degradation of Hypomodified Initiator tRNA\u3csup\u3eMet\u3c/sup\u3e in \u3cem\u3eS. cerevisiae\u3c/em\u3e

The tRNA m1A58 methyltransferase is composed of two subunits encoded by the essential genes TRM6 and TRM61 (formerly GCD10 and GCD14). The trm6-504 mutation results in a defective m1A methyltransferase (Mtase) and a temperature-sensitive growth phenotype that is attributable to the absence of m1A58 and consequential tRNAiMet instability. We used a genetic approach to identify the genes responsible for tRNAiMet degradation in trm6 cells. Three recessive extragenic mutations that suppress trm6-504 mutant phenotypes and restore hypomodified tRNAiMet to near normal levels were identified. The wild-type allele of one suppressor, DIS3/RRP44, encodes a 3′-5′ exoribonuclease and a member of the multisubunit exosome complex. We provide evidence that a functional nuclear exosome is required for the degradation of tRNAiMet lacking m1A58. A second suppressor gene encodes Trf4p, a DNA polymerase (pol σ) with poly(A) polymerase activity. Whereas deletion of TRF4 leads to stabilization of tRNAiMet, overexpression of Trf4p destabilizes the hypomodified tRNAiMet in trm6 cells. The hypomodified, but not wild-type, pre-tRNAiMet accumulates as a polyadenylated species, whose abundance and length distribution both increase upon Trf4p overexpression. These data indicate that a tRNA surveillance pathway exists in yeast that requires Trf4p and the exosome for polyadenylation and degradation of hypomodified pre-tRNAiMet

Polyadenylation Dependent Nuclear RNA Surveillance in Saccharomyces Cerevisiae

Cells are composed of several types of RNAs such as messenger RNA (mRNA), transfer RNA (tRNA), small nuclear and small nucleolar RNAs (sn and snoRNAs) and ribosomal RN As (rRNAs) that perform various cellular activities like protein synthesis. tRNA serves as an adaptor in protein synthesis by transferring a specific amino acid to the site of protein synthesis. A newly synthesized tRNA consists of a 5\u27 leader and 3\u27 trailer which are removed during processing to generate mature tRNA. During its biogenesis, aberrant tRNAs might be produced due to processing errors and if not degraded they might get incorporated in to the protein synthesis machinery forming abnormal proteins. Hence degradation mechanisms for RNAs have evolved to ensure normal functioning of cells. One of the striking features of a tRNA molecule is the presence of numerous post transcriptional modifications. Methylation of adenosine at position 58 is a post transcriptional modification in tRNA that is catalyzed by the enzyme complex Trm6p/Trm6lp in yeast, Saccharomyces cerevisiae. In TRM6 and TRM61 mutants, tRNAs lack m1A modification and this renders a unique tRNA, Initiator tRNA Met (tRNAiMet) unstable and targets it for degradation. I have characterized the molecular details of this novel degradation pathway of hypomethylated tRNAiMet. Genetic suppressor analysis of a trm6-504 mutant showed that RRP44, a subunit of a multiprotein RNA processing and degradation complex, exosome and TRF4, a member of nucleotidyltransferase family of proteins are required for the degradation of hypomethylated tRNAiMet. Deletion of another exosome subunit gene, RRP6, impaired degradation ofhypomethylated tRNAiMet proving strong evidence that the exosome is required for the degradation of tRNAte\u27. As Rrp6p is associated only with the nuclear form of the exosome, I conclude that bulk of the degradation ofhypomethylated tRNAiMet in trm6-504 cells occurs in the nucleus..

Polyadenylation dependent nuclear RNA surveillance in Saccharomyces cerevisiae

Cells are composed of several RNAs such as messenger RNA (mRNA), transfer RNA (tRNA), small nuclear and small nucleolar RNAs (sn and snoRNAs) and ribosomal RNAs (rRNAs) that perform various cellular activities such as protein synthesis. tRNA serves as an adaptor in protein synthesis as it transfers a specific amino acid to the site of protein synthesis. A newly synthesized tRNA consists of a 5\u27 leader and 3\u27 trailor which are removed to generate mature tRNA. During biogenesis, aberrant tRNAs might be produced due to processing errors and if not degraded they might get incorporated in to the protein synthesis machinery forming abnormal proteins. Thus degradation mechanisms for RNAs have evolved to ensure normal functioning of cells. One of the striking features of a tRNA molecule is the presence of numerous post transcriptional modifications one of which is Methylation of adenosine at position 58 of tRNA catalyzed by the enzyme complex Trm6p/Trm61p in yeast, Saccharomyces cereviseae . In TRM6 and TRM61 mutants, tRNAs lack m1 A modification and this results in the degradation of a unique tRNA, Initiator tRNAMet (tRNA iMet ). I have characterized the molecular details of this novel degradation pathway of hypomethylated tRNAiMet . Genetic suppressor analysis of a trm6-504 mutant showed that RRP44 , a subunit of a multiprotein RNA processing and degradation complex, the exosome and TRF4 , a member of polβ nucleotidyl transferase family of proteins are required for the degradation of hypomethylated tRNAiMet which occurs in the nucleus. The in vivo substrate for polyadenylation and degradation is the precursor form of hypomethylated tRNAi Met which possesses the unprocessed 5\u27 and 3\u27 extensions, demonstrating that the integrity of tRNA is tested early during its biogenesis. Interestingly, Trf5p, homolog of Trf4p is required for efficient degradation of tRNA iMet . Nuclear tRNA surveillance is a global mechanism to degrade aberrant RNAs as mutant U6 SnRNA and 5S rRNA are polyadenylated and degraded by Trf4p and the exosome similar to hypomethylated tRNA iMet . This study has initiated a new area of study, RNA surveillance that involves polyadenylation by Trf4p and degradation by the exosome

Nuclear RNA surveillance in Saccharomyces cerevisiae: Trf4p-dependent polyadenylation of nascent hypomethylated tRNA and an aberrant form of 5S rRNA

1-Methyladenosine modification at position 58 of tRNA is catalyzed by a two-subunit methyltransferase composed of Trm6p and Trm61p in Saccharomyces cerevisiae. Initiator tRNA (tRNA(i)(Met)) lacking m(1)A58 (hypomethylated) is rendered unstable through the cooperative function of the poly(A) polymerases, Trf4p/Trf5p, and the nuclear exosome. We provide evidence that a catalytically active Trf4p poly(A) polymerase is required for polyadenylation of hypomethylated tRNA(i)(Met) in vivo. DNA sequence analysis of tRNA(i)(Met) cDNAs and Northern hybridizations of poly(A)+ RNA provide evidence that nascent pre-tRNA(i)(Met) transcripts are targeted for polyadenylation and degradation. We determined that a mutant U6 snRNA and an aberrant form of 5S rRNA are stabilized in the absence of Trf4p, supporting that Trf4p facilitated RNA surveillance is a global process that stretches beyond hypomethylated tRNA(i)(Met). We conclude that an array of RNA polymerase III transcripts are targeted for Trf4p/ Trf5p-dependent polyadenylation and turnover to eliminate mutant and variant forms of normally stable RNAs

Nuclear surveillance and degradation of hypomodified initiator tRNA(Met) in S. cerevisiae

The tRNA m(1)A58 methyltransferase is composed of two subunits encoded by the essential genes TRM6 and TRM61 (formerly GCD10 and GCD14). The trm6-504 mutation results in a defective m(1)A methyltransferase (Mtase) and a temperature-sensitive growth phenotype that is attributable to the absence of m(1)A58 and consequential tRNA(i)(Met) instability. We used a genetic approach to identify the genes responsible for tRNA(i)(Met) degradation in trm6 cells. Three recessive extragenic mutations that suppress trm6-504 mutant phenotypes and restore hypomodified tRNA(i)(Met) to near normal levels were identified. The wild-type allele of one suppressor, DIS3/RRP44, encodes a 3′-5′ exoribonuclease and a member of the multisubunit exosome complex. We provide evidence that a functional nuclear exosome is required for the degradation of tRNA(i)(Met) lacking m(1)A58. A second suppressor gene encodes Trf4p, a DNA polymerase (pol σ) with poly(A) polymerase activity. Whereas deletion of TRF4 leads to stabilization of tRNA(i)(Met), overexpression of Trf4p destabilizes the hypomodified tRNA(i)(Met) in trm6 cells. The hypomodified, but not wild-type, pre-tRNA(i)(Met) accumulates as a polyadenylated species, whose abundance and length distribution both increase upon Trf4p overexpression. These data indicate that a tRNA surveillance pathway exists in yeast that requires Trf4p and the exosome for polyadenylation and degradation of hypomodified pre-tRNA(i)(Met)

Additional file 4: of OSM potentiates preintravasation events, increases CTC counts, and promotes breast cancer metastasis to the lung

Figure S3. Deterioration of physical condition in MDATO/OSM tumor-bearing mice treated with TET. a MDATO/OSM tumor-bearing mice treated with tetracycline (+TET) lost, on average, 11.4% of their body weight during TET treatment, compared with −TET mice, which gained an average of 5.5% of their body weight over the same period. b Representative image of mice with MDATO/OSM tumors +TET shows prominent spinal column, muscle wasting, and lack of visible adipose tissue. c Gross morphology of normal (left) and abnormal kidneys (right). Normal kidneys have a distinct border between the medulla and the cortex, with the cortex shown in a darker pink/red color and the medulla shown in a lighter pink color. This indicates that normal blood perfusion was taking place. Abnormal kidneys were either both pale and hypoperfused (middle) or damaged (right), with no clear distinction between the cortex and the medulla. d One hundred percent of mice in the +TET group have abnormal kidney morphologies, whereas only 25% of the mice in the −TET group have abnormal kidneys. **p < 0.01 by Fisher’s exact test. e Sera from mice with abnormal kidneys have a statistically significant higher level of OSM than sera from mice with normal kidneys. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 by two-tailed Student’s t test. (ZIP 135 kb

Additional file 2: of OSM potentiates preintravasation events, increases CTC counts, and promotes breast cancer metastasis to the lung

Figure S1. qPCR standard curve derived from spiking cancer cells into mouse blood. MDA-MB-231 cells were spiked into mouse blood, and DNA was extracted and subjected to qPCR analysis. Specific cell numbers were correlated to CT values and were used to construct a standard curve for the CT values extrapolated from experimental mouse blood. (PPTX 186 kb

Additional file 3: of OSM potentiates preintravasation events, increases CTC counts, and promotes breast cancer metastasis to the lung

Figure S2. Representative OSM staining intensity in IHC. 0 = no staining (image not shown), 1 = light staining, 2 = moderate staining, 3 = heavy staining. (PPTX 27 kb

Additional file 8: of OSM potentiates preintravasation events, increases CTC counts, and promotes breast cancer metastasis to the lung

Figure S7. Test of cell line-specific variance in colony-forming assay between 4T1.2-shLacZ and 4T1.2-shOSM2 cell lines. Approximately 10 and 50 cells of 4T1.2-shLacZ or 4T1.2-shOSM2 cells were seeded onto tissue culture plates and were allowed to incubate until colony formation. No significant differences between the cells were detected with ~ 10 cells seeded; however, there was a small but significant increase in the number of colonies with 4T1.2-shOSM2 cells at 50 cells seeded. Data are expressed as mean ± SEM. *p < 0.05 by one-way ANOVA with Bonferroni’s multiple comparisons test. (PPTX 68 kb