Stable PNPase RNAi silencing: its effect on the processing and adenylation of human mitochondrial RNA

Abstract

Polynucleotide phosphorylase (PNPase) is a diverse enzyme, involved in RNA polyadenylation, degradation, and processing in prokaryotes and organelles. However, in human mitochondria, PNPase is located in the intermembrane space (IMS), where no mitochondrial RNA (mtRNA) is known to be present. In order to determine the nature and degree of its involvement in mtRNA metabolism, we stably silenced PNPase by establishing HeLa cell lines expressing PNPase short-hairpin RNA (shRNA). Processing and polyadenylation of mt-mRNAs were significantly affected, but, to different degrees in different genes. For instance, the stable poly(A) tails at the 3' ends of COX1 transcripts were abolished, while COX3 poly(A) tails remained unaffected and ND5 and ND3 poly(A) extensions increased in length. Despite the lack of polyadenylation at the 3' end, COX1 mRNA and protein accumulated to normal levels, as was the case for all 13 mt-encoded proteins. Interestingly, ATP depletion also altered poly(A) tail length, demonstrating that adenylation of mtRNA can be manipulated by indirect, environmental means and not solely by direct enzymatic activity. When both PNPase and the mitochondrial poly(A)-polymerase (mtPAP) were concurrently silenced, the mature 3' end of ND3 mRNA lacked poly(A) tails but retained oligo(A) extensions. Furthermore, in mtPAP-silenced cells, truncated adenylated COX1 molecules, considered to be degradation intermediates, were present but harbored significantly shorter tails. Together, these results suggest that an additional mitochondrial polymerase, yet to be identified, is responsible for the oligoadenylation of mtRNA and that PNPase, although located in the IMS, is involved, most likely by indirect means, in the processing and polyadenylation of mtRNA.

FIGURE 1.

Characterization of HeLa cell lines with stable shRNA-mediated PNPase-silencing. (A) Western and Northern blots showed substantial silencing of PNPase in three PNPase-shRNA clonal cell lines; E1, E3, and G3, compared to non-transfected HeLa cells (wt), and a negative control line termed “empty vector” (EV). (B) Proliferation assay in which identical amounts of cells from each line were plated, counted after 96 h, and the resulting number divided by the initial amount, yielding the “fold increase” rate (FI). (C) Mitochondrion-encoded proteins were labeled by incubating cells with [³⁵S]methionine–[³⁵S]cysteine in the presence of the 80S ribosome inhibitor, emetine. Following radioactive labeling for 25 min to measure protein translation and 120 min to measure accumulation, in left and right gel pictures, respectively, equal amounts of extracted proteins were fractionated on SDS-PAGE, dried, and analyzed by autoradiography. Identification of the 13 mitochondrion-encoded proteins is presented to the right. A panel from the gels stained with Coomassie (Comm.) is included below. (D) Oligo(dT) RT-PCR of the COX1 mRNA in the different cell lines is presented in the upper part. Relative amounts of the transcript were determined by nonsaturating PCR amplification of oligo(dT)-primed cDNA with gene-specific primers and fractionation on agarose gel. Quantitative control was achieved by β-actin PCR amplification using randomly primed cDNA. RNA blots analysis is presented in the lower part. Total RNA extracted from the cell lines was fractionated by denaturing agarose gels, transferred to nylon membrane, and hybridized with ³²P-specific oligo-DNA probe for the COX1 and β-actin (used as loading control) transcripts.

FIGURE 2.

Improper polyadenylation and processing of the COX1 transcript in PNPase-silenced cell lines. (A) cRT-PCR labeling and sequencing methods, used to investigate the 5′ and 3′ ends of a target mRNA, are described. Both start with the circularization of total RNA which contains the target mRNA, with T4 RNA ligase. Next, a gene-specific reverse oligo, generally termed R1, is used to prime reverse transcription, initiated ∼100 nt downstream of the 5′ end. Afterward, two consecutive PCR reactions with F1+R2 and F2+R2 oligos, respectively, amplify the adjoined 5′ and 3′ extremities and simultaneously increase specificity. At this point, there are two options: For sequencing, the products are cloned to T/A vectors, PCR-screened, and sequenced, in order to analyze individual clone sequences (cRT-PCR sequencing). To obtain a more global view of the target mRNA population instead, a third PCR reaction, similar to the second, can be applied, in which either the R2 or F2 oligo is labeled with [γ-³²P]ATP. Products are resolved in 10% acrylamide gel, followed by autoradiography (cRT-PCR labeling). The 3′ poly(A) tail lengths can be calculated by subtracting the expected length of a properly processed naked 3′ end molecule from that of the actual product as compared to a nucleotide ladder. (B) The 3′ and 5′ ends of COX1 were analyzed in control (wt and EV) and PNPase-silenced (E1, E3, and G3) cells using the cRT-PCR labeling technique (as described above for A). Products were resolved by 10% denaturing PAGE, followed by autoradiography, and product size was determined by comparison to a nucleotide ladder produced by alkaline hydrolysis of a [³²P]RNA (lane M). Assuming proper processing of the mRNA, the product size represents the length of the poly(A) tail added to the 3′ end, a naked 3′ end marked as “0.” However, products could also originate from molecules with impaired processing. In order to differentiate between these two possibilities, cRT-PCR sequencing was performed as shown in part C of the figure. (C) cRT-PCR sequencing of COX1 is shown. The region of the human mitochondrial genome containing the COX1 gene is schematically displayed at the bottom. The first nucleotide of the COX1 transcript at the 5′ end is marked as +1. The translation initiation codon starts at number +4, and the amino acid coding region is colored in dark gray with the two diagonal lines indicating that it is not drawn to scale. The 5′ and 3′ UTRs, composed of 3 nt and the tRNA^K antisense, respectively, are shown in light gray. The flanking sequences, including the 9-nt intergenic region and tRNA^Y antisense located upstream of the COX1 gene, are marked with a dashed white line. Four black arrows represent the R2, R1, F2, and F1 primers used in cRT-PCR. Above the gene scheme, individually sequenced COX1 clones are shown for each cell line. A dashed line symbols the inferred internal part of the COX1 mRNA that was not physically isolated, as only the transcript extremities were amplified (as described above for A). Black lines show the sequenced segments of the 5′ and 3′ ends with the relative position aligned to the scheme below. The 5′ end sites, initiating at positions other than the proper +1, are labeled in parentheses. At the 3′ end of the transcript, either the number of adenosines is indicated or, in parentheses, the post-transcriptionally added nonadenosine extensions that could be located either at the 3′ or at the 5′ end of the transcript.

FIGURE 3.

Varied effects of PNPase-silencing on the stable polyadenylation of several mitochondrial transcripts. (A) The 3′ ends of ND5, ND3, and COX3 were analyzed by cRT-PCR labeling in control and PNPase-silenced cells. Labels are the same as in Fig. 2B. The oligo(A) and poly(A) fractions are indicated to the right of each gel. Note that, for ND5 and ND3, the poly(A) fractions in E3 and G3 cells were extended by several nucleotides and in E1, significantly diminished. (B) cRT-PCR sequencing of 16S rRNA did not detect an effect of PNPase knockdown on the nature of 3′ oligoadenylation of this transcript. Gene scheme labels are the same as in Fig. 2C.

FIGURE 4.

Truncated nonabundant COX1 transcripts with adenosine tails were isolated from cells with either PNPase or mtPAP silencing. (A) Transient mtPAP RNAi was applied to wild-type cells as well as PNPase-silenced lines, and mtPAP mRNA levels were measured with semiquantitative RT-PCR pre- (five left lanes) and post- (five right lanes) mtPAP siRNA transfection. PNPase silencing did not affect mtPAP levels, but application of mtPAP siRNA successfully lowered mtPAP mRNA levels to less than 10%. (B) The oligo(dT)-RT-PCR method was used to isolate nonabundant, truncated, polyadenylated mitochondrial transcripts, assumed to be degradation intermediates. RT was primed with an oligo(dT)-adapter and product cDNA was subjected to a PCR reaction with a gene-specific forward primer, generally termed F1, paired with the adapter oligo. Resulting products were used as templates for a second round of PCR with a nested forward primer, termed F2, paired with the adapter oligo and resulting products were then cloned. Primer F3 was paired with the adapter-oligo to isolate the positive clones which were then sequenced. The schematic presentation of the COX1 gene is as in Fig. 2C. (C) The left table summarizes the effect of either PNPase or mtPAP RNAi-mediated silencing on the adenylation of truncated COX1 fragments, believed to be degradation intermediates. These molecules were isolated with the oligo(dT)-RT-PCR method, as described in the scheme (see B). PNPase silencing did not affect tail length, while knockdown of mtPAP resulted in a decrease in tail length. In the graph to the right, the tail lengths of each individual sequenced clone are presented and compared between assays 1, 2, and 3, as described in the table.

FIGURE 5.

cRT-PCR labeling and sequencing assays disclosed the effect of mtPAP silencing on the polyadenylation of the mature 3′ ends of ND5 and ND3. (A) Transient mtPAP RNAi was applied to wt cells as well as PNPase-silenced lines, as shown in Fig. 4A. RNA purified from these cells and also from a non-transfected control was then subjected to cRT-PCR labeling to analyze ND3. The oligo(A) and poly(A) fractions are indicated to the right of the gel. The poly(A) fraction was barely detectable. However, the oligo(A) fraction intensified substantially, even in cells with concurrent stable PNPase and transient mtPAP silencing. (B) ND5 cRT-PCR labeling as described for A. The poly(A) fraction was hardly detectable, and, unlike ND3, no oligo(A) tails accumulated; instead, only molecules with naked 3′ ends were present. (C) Results of cRT-PCR sequencing for ND5 in control (wt and EV), and PNPase knockdown lines (E1, E3, and G3) are shown along with those of transient silencing of mtPAP in wt and E3 cells (upper 16 lines). Statistically, no difference could be seen using the cRT-PCR sequencing method, between control (wt and EV) and the PNPase-silenced lines, despite the effect revealed in the corresponding cRT-PCR labeling assay (Fig. 3A). However, as disclosed in the cRT-PCR labeling assay (see B), the majority of transcripts from mtPAP siRNA-transfected cells (upper 16 lanes) lacked oligo(A) extensions. The ND5 gene scheme and sequenced clone labels are the same as for COX1, as in Fig. 2C.

FIGURE 6.

PNPase silencing caused changes in cellular ATP level and membrane potential in PNPase-silenced cells, and ATP depletion manipulated poly(A)-tail length. (A) Immunoblot analysis, of proteins obtained from control (mock transfected) cells and cells transiently transfected with PNPase siRNA duplexes, is shown. (B) ATP levels were determined in the stable PNPase-silenced cell lines before and after additional transient PNPase siRNA transfection. Levels were normalized by total protein and are presented as percentages of wt ATP. The measurement for cells in which ATP had been depleted with azide and deoxyglucose is shown at the far right. (C) Mitochondrial membrane potential (Δψ) was measured in the five cell lines using the cationic indicator tetramethylrhodamine methyl ester (TMRM). The Δψ was determined by subtracting the TMRM fluorescence at mitochondrial saturation (minimum) from the fluorescence detected upon addition of the uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP) (maximum). The results of one representative experiment are shown to the left and a table summarizing multiple measurements in which the results are presented as percentages of wt Δψ is shown to the right. (D) cRT-PCR labeling assays for COX1 and ND3 were applied to RNA from mock siRNA control and transient PNPase siRNA cells to compare the effect of transient silencing to that of stable (four left lanes). Note the elongation of the COX 1 and ND3 poly(A) fraction in transient PNPase RNAi. To assess the effect of ATP depletion on mtRNA adenylation, cRT-PCR labeling was applied to control and ATP depletion cells, for ND3 and ND5 (four right lanes). Although ATP levels decreased in PNPase-silenced cells, as shown in B, and poly(A) tails lengthened, ATP depletion with azide and deoxyglucose caused the opposite effect; shortening of the poly(A) tail fraction in both genes and, in ND5, shortening of the oligo(A) tails as well.