Search for antisense copies of beta-globin mRNA in anemic mouse spleen

Abstract

Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS) globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

Background

Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS) globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences.

Results

We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%.

Conclusions

Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

Background

], many of the claims have been substantiated and in a subset of these, biological significance has been established. The origin of most of these AS sequences is transcription via DNA-directed RNA polymerization.

].

], but apparently there is no involvement of RNA-directed RNA polymerase activity.

].

Induction of splenomegaly by injections with phenylhydrazine

It involved 6 daily injections of PHZ into the peritoneal cavity.

, they only reported results for samples taken at a single time, day 8. However, since we were interested in the possibility that the status of the animal in this response might be important, we considered animals taken before (i.e., untreated control mice), during, and following the PHZ treatments.

, the mass of the mouse spleen increased 5-fold even by day 5. This splenomegaly was maintained for at least 3 days after the injections were halted. After 10 days (i.e., 15 days after the PHZ injections were begun) the spleen was reduced in size but was still 2-times bigger than the mass prior to day 0, the time of initiation of the PHZ injections.

Induction of splenomegaly by treatment of mice with phenylhydrazine. Mice were subjected to i.p. injections of phenyhydrazine (PHZ) solution at the times as indicated by arrows in the figure. Subsequently animals were euthanized and the spleens removed and weighed, as indicated. Each circular symbol represents a single mouse.

Northern analyses to detect 600 nt AS globin RNA

Clearly, as a consequence of PHZ treatment, the spleen promptly underwent a major increase in mass that was followed by a return to almost normal size. To explore the possibility that some form of antisense RNA with or without PTGS, was involved either during or after treatment, we examined RNA samples taken at a series of times.

), we deduce that the level of accumulation of β-globin mRNA per spleen was increased about 30-fold as a consequence of the PHZ treatment.

transcribed globin AS RNA (lane 1), globin sense RNA (lane 2), and double-stranded globin cDNA (lane 3). As expected, these three standards exhibit mobilities somewhat faster than that of mature globin mRNA. In all panels lanes 4-9 represent RNA from spleens of mice at 0, 5, 8, 10, 12, and 15 days, respectively, after the beginning of the PHZ injections. As size standards we used 5'-labelled DNA fragments, with sizes indicated at the left of each panel.

This greatly increased level of β-globin mRNA in the spleen was probably attributable to multiple factors, e.g., increased rates of transcription could be one of the factors, but there is no doubt that the number of reticulocytes in the spleen was increased under these conditions.

The discrepancy may have arisen because of differences in hybridization conditions, such as stringency; unfortunately, we were unable to find the conditions of hybridization used by those authors.

, lanes 4-9).

). For all six spleen samples (lanes 4-9) the signal of 28 S rRNA per fixed amount of total RNA was essentially constant.

]. Second, and of more significance, not only were the signal strengths of putative antisense relative to sense globin the same (about 3%) for each of the 6 spleen samples (lanes 4-9, panels C and D) but the same ratio was obtained for the standards of antisense and sense standards, that were synthesized in vitro. In other words, most, if not all, of the signal that appeared as AS RNA in the splenic samples was explainable as an artifact of hybridization.

Overall, the two main conclusions from these northern analyses are first, that no AS to globin RNA was detected with oligonucleotide probes and second, that the signal detected with a full-length RNA probe was, at least predominantly, due to an artifact of hybridization.

Discussion

).

].

). However, as described in the results, we can conclude that the signals detected were at least predominantly due to a hybridization artifact. One interpretation of this artifact might be that it arose because of the intramolecular base-pairings in the globin mRNA. This would allow that even radioactive sense sequences might make interactions in a northern analysis with unlabeled sense RNA, even under conditions (hybridization and subsequent washing at 65°C) that we would consider stringent. However, a more likely explanation is that as an artifact of in vitro transcription, the radioactive RNA probes also contain trace amounts of the opposite strand; thus, when used in hybridization in the absence of appropriate specificity controls, one might detect species that would be incorrectly interpretted as AS RNA species.

Anemic mice

]. Each injection was of 0.1 ml of 0.8% neutralized PHZ in 0.015 M sodium acetate. At days 5, 8, 10, 12, and 15, animals were euthanized and the spleens promptly removed and weighed. Aliquots of 50-100 mg were then incubated at 4°C in a solution of RNAlater (Ambion), after which the liquid was removed and the samples frozen at -80°C.

RNA extraction

Frozen samples of spleen tissue were added directly into 10 volumes of Tri-Reagent (Molecular Research Center) followed by immediate processing using a Brinkmann homogenizer. We then followed the manufacturer's instructions to collect the RNA free of DNA and protein.

Plasmids and RNA transcription

with phage T7 RNA polymerase (Promega). Similarly, to make AS RNAs the construct was digested with EcoRI and copied with phage T3 RNA polymerase (Life Sciences). From this construct we were also able to release the double-stranded cDNA insert, for use as a hybridization standard for normalizing the hybridizations to detect sense and AS strands of globin sequence. In order to detect glyceraldehyde-3-phosphate dehydrogenase (GAPDH) we used a construct with a 1 kb EcoRI cDNA fragment inserted into pBS SK- (Stratagene) to make an RNA probe.

Northern analyses

, the filter was hybridized with an RNA probe specific for mouse GAPDH mRNA.

Abbreviations

AS, antisense; PHZ, phenylhydrazine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Commentary

This paper was accepted for publication dependent on its association with the following commentary from one of the peer reviewers. The commentary can be accessed here:

Acknowledgments

This work was supported by grants AI-26522 and CA-06927 from the N.I.H., and by an appropriation from the Commonwealth of Pennsylvania. Anthony Lerro and Jackie Valvardi performed the animal experiments. Maureen Murphy provided the GAPDH clone. Finally, constructive comments on the manuscript were provided by Glenn Rall, Richard Katz, Jinhong Chang, and Gloria Moraleda.