Role of RNase MRP in viral RNA degradation and RNA recombination

Abstract

RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

FIG. 1.

Model of the roles of endo- and exoribonucleases in tombusvirus RNA degradation and recombination. During or after replication of TBSV DI-72 repRNA, a host endoribonuclease cleaves some of the viral RNAs (at places marked by arrows), producing 5′Fr and 3′Fr degRNAs. These cleaved repRNA products are then rapidly removed by the host Xrn1p 5′-3′ exoribonuclease. The rapid removal of the recombination substrates inhibits viral RNA recombination. The not-yet-degraded 3′Fr RNAs can participate in replicase-driven recombination events. Note that 5′Fr lacks important cis-acting replication sequences and does not seem to participate in RNA recombination.

FIG. 2.

Downregulation of Snm1p level inhibits the accumulation of TBSV degRNA in yeast lacking Met22p bisphosphate-3′-nucleotidase. The met22Δ yeast strain shows decreased Xrn1p activity, facilitating the detection of endoribonucleolytically cleaved TBSV repRNA products. (A) Northern blot analysis for detection of plus strands of TBSV DI-72 repRNA and the newly formed degRNAs and recRNAs from met22Δ yeast strains. The accumulating repRNA, degRNA, and recRNA are shown with arrows. The numbers at the bottom of the panel show percentages of degRNA2 accumulation normalized to the level of DI-72 repRNA (100% in each sample). The various 5′-to-3′ sequences present in the repRNA and the generated recRNAs and degRNAs are shown on the right. The data were obtained from 24 independent yeast streaks. (B) Ethidium bromide-stained gel image showing partial processing of 5.8S rRNA in yeast with downregulated Snm1p. (C) Western blot image showing reduced expression level of p33 replication protein in yeast with downregulated Snm1p.

FIG. 3.

Increased accumulation of TBSV recRNAs in yeast overexpressing plasmid-borne NME1 RNA. (A) Northern blot analysis of total RNA samples obtained from the shown yeast strains overexpressing wt NME1 RNA from a plasmid. The accumulating repRNA, degRNA, and recRNAs are shown with arrows. The numbers at the bottom of the panel show percentages of degRNA2, recRNA1, and recRNA2 accumulation normalized to the level of DI-72 repRNA (100% in each sample). The various sequences present in the (+)repRNA and the newly formed recRNAs are shown schematically on the right. (B) Northern blot analysis of total RNA samples obtained from yeast strain BY4741, with a normal level of NME1 RNA expression from the chromosome (lane 1) or overexpressing wt NME1 RNA from a plasmid and from the chromosome (lanes 2 to 4).

FIG. 4.

Reduced accumulation of TBSV degRNA in yeast expressing nme1^ts RNA from a plasmid. (A) Northern blot analysis of total RNA samples obtained from nme1Δ yeast expressing plasmid-borne wt NME1 RNA or a temperature-sensitive nme1^ts RNA at the semipermissive temperature of 25°C. The accumulating repRNA, degRNA, and recRNAs are shown with arrows. The numbers at the bottom of the panel show percentages of degRNA2, recRNA1, and recRNA2 accumulation normalized to the level of DI-72 repRNA (100% in each sample). The various sequences present in the (+)repRNA and the newly formed recRNAs are shown schematically on the right. (B) Ethidium bromide-stained gel image showing the accumulation of repRNA. (C) Western blot image showing the expression level of p33 replication protein in the above yeast strains.

FIG. 5.

Increased accumulation of TBSV repRNA and decreased level of degRNAs in yeast expressing NME1 or nme1^ts RNA from a plasmid at the semipermissive temperature of 34°C. (A) Northern blot analysis of total RNA samples obtained from nme1Δ yeast cultured at 34°C. The yeast cells expressed plasmid-borne wt NME1 or nme1^ts RNA. The accumulating repRNA and degRNA-RI were detected with an RI probe that does not detect recRNAs. The numbers at the bottom of the panel show percentages of repRNA and degRNA-RI accumulation (the latter was normalized to the level of DI-72 repRNA). (B) Northern blot analysis of the above total RNA samples, using an RIII/RIV probe that also detects recRNAs, in addition to repRNA and degRNAs. The bottom image shows a Northern blot of the above total RNA samples, using a 5S rRNA probe as a loading control. Note that the level of repRNA was normalized based on the loading control. (C) Western blot image showing expression level of the p33 replication protein in the above yeast strains.

FIG. 6.

Partial inactivation of RNase MRP decreases formation of degRNAs and increases accumulation of repRNA in yeast. (A) Relative accumulation levels of three different degRNAs in nme1Δ xrn1Δ yeast (expressing the plasmid-borne nme1^ts RNA) cultured at either the permissive temperature of 20°C or the semipermissive temperature of 34°C (gray bars). A comparable analysis was performed with degRNAs in nme1Δ xrn1Δ yeast expressing the plasmid-borne wt NME1 RNA, which is not temperature sensitive (black bars). The measurements are based on Northern blot analysis of total RNA samples obtained from the above nme1Δ yeast with either an RI probe or an RIV probe. Note that the above yeast strain lacks Xrn1p exoribonuclease to facilitate the detection of endoribonucleolytically cleaved degRNAs. (B) Relative accumulation of TBSV DI-72 repRNA in nme1Δ xrn1Δ yeast expressing plasmid-borne wt or nme1^ts (TS) RNA. The yeast cells were cultured at either the permissive temperatures of 20°C and 23°C or the semipermissive temperatures of 30°C and 34°C. The measurements are based on Northern blot analysis of total RNA samples obtained from the above nme1Δ xrn1Δ yeast with an RIV probe. Note that the level of repRNA was normalized based on the 5S rRNA probe as a loading control.

FIG. 7.

In vitro endoribonucleolytic cleavage of TBSV DI-72 (+)repRNA by RNase MRP. (A) Increasing amounts of highly purified RNase MRP were added to internally labeled DI-72 RNA, followed by denaturing PAGE analysis. The bands likely representing DI-ΔRI and RI, which are due to endoribonucleolytic cleavage of the internally labeled DI-72 RNA, are marked. To test the origins of the different endoribonucleolytic cleavage products, we used RI (B) and RIV (C) probes and unlabeled repRNAs. Note that in addition to the full-length DI-72 RNA, we also used truncated derivatives lacking one of the four regions of DI-72, as indicated by the name of the given RNA. Asterisks indicate the input-sized RNAs (not visible are those input RNAs which had deletion of the region targeted by the probe), while the endoribonucleolytic cleavage product representing RI is marked with a filled arrowhead.

FIG. 8.

In vitro endoribonucleolytic cleavage of TBSV genomic (+)RNA by RNase MRP. (A) Increasing amounts of highly purified RNase MRP were added to unlabeled TBSV gRNA, followed by primer extension and denaturing PAGE analysis. The primer extension was performed with labeled 708R primer. Bands representing mapped endoribonucleolytically cleaved products are marked with arrows. We used a 5′-phosphorylated 100-bp ladder (NE Biolabs) and a 25-bp ladder (Gibco-BRL) as markers. (B) Similar cleavage-site mapping experiment to that in panel A, except that the primer extension was performed with labeled RIII (3820) primer. (C) Locations of mapped endoribonucleolytic cleavage sites in TBSV gRNA. Note that we show only the more frequent/repeatable endoribonucleolytic cleavage sites and their approximate locations.

FIG. 9.

Role of RNase MRP in TBSV RNA degradation and RNA recombination in plants. (A) (Left) Phenotype of 7-2 RNA/NME1 knockdown N. benthamiana plants 14 days after agroinfiltration with TRV silencing vectors. (Right) Effect of knockdown of 7-2 RNA/NME1 on symptoms caused by CNV infection in N. benthamiana 8 and 10 days after the second agroinfiltration. VIGS was performed via agroinfiltration of vector (TRV) carrying the 7-2 RNA/NME1 sequence or the TRV empty vector (as a control). Coagroinfiltration to express TBSV DI-72 repRNA together with CNV gRNA was done 9 days after silencing of 7-2 RNA/NME1 expression by agroinfiltration. (B) Northern blot analysis of CNV gRNA accumulation in the agroinfiltrated leaves of 7-2 RNA/NME1 knockdown or control N. benthamiana plants 4 days after the second agroinfiltration. rRNA (not shown) was used as a loading control. The plant leaf samples were obtained from 5 separate experiments (total of 5 × 48 samples). (C) Decreased accumulation of TBSV degRNA-RI in 7-2 RNA/NME1 knockdown N. benthamiana plants compared to that in the control plants, which were agroinfiltrated with the TRV empty vector, 4 days after the second agroinfiltration. The accumulation level of degRNA-RI was normalized to the level of DI-72 repRNA. Note that the RI probe was specific for the TBSV repRNA and that the helper CNV gRNA or its degradation products were not detected. (D) Northern blot analysis of TBSV repRNA and newly formed recRNAs in agroinfiltrated leaves of 7-2 RNA/NME1 XRN4 double-knockdown or XRN4 single-knockdown control N. benthamiana plants. The numbers at the bottom of the panel show percentages of recRNA2 and recRNA3 accumulation normalized to the level of DI-72 repRNA (100% in each sample). rRNA (not shown) was used as a loading control. Note that the RIV probe was specific for the TBSV repRNA and that the helper CNV gRNA or its degradation products were not detected. (E) Decreased accumulation of TBSV degRNA-RI in 7-2 RNA/NME1 XRN4 double-knockdown plants compared to that in the control XRN4 single-knockdown N. benthamiana plants. See further details above as described for panel C.