Biochemical basis of the RPM
Although PMY has been extensively used for decades (Pestka, 1971
; Prouty et al., 1975
), the possible use of puromycylation as a method of identifying translating ribosomes in situ has not been previously explored. We reasoned that by briefly pretreating cells with translation elongation inhibitors like CHX or emetine (Pestka, 1971
), we could freeze translation and then “puromycylate” immobilized nascent chains by incubating cells with PMY and detect actively translating ribosomes in permeabilized cells or cell extracts using the PMY-specific mAb 12D10 (Schmidt et al., 2009
) to PMY tethered to ribosomes by a nascent chain ().
Figure 1. Characterizing the RPM biochemically. (A) Schematic representation of the RPM. After freezing polysomes with an elongation inhibitor (step 1), PMY is added (step 2) to living cells or subcellular fractions, and nascent chains are puromycylated through (more ...)
Despite CHX pretreatment nearly completely blocking translation as measured by incorporation of [35
S]methionine into acid-insoluble proteins (Fig. S1 A
), its continued presence has no significant effect on nascent chain puromycylation after 5-min exposure to PMY, as detected by anti-PMY immunoblotting of total cell lysates (). Emetine, an irreversible and highly effective translation elongation inhibitor, actually enhances puromycylation (), likely because of some combination of greater effectiveness (it is irreversible) and lack of interference with PMY association with ribosomes (Pestka et al., 1972
). Consequently, we preferentially used emetine for the RPM. In contrast, blocking translation initiation with pactamycin (an initiation inhibitor) or arsenite (an oxidizing stressor) inhibits puromycylation, which we attribute to the release of nascent chains as their translation is completed, generating nontranslating monosomes (). Importantly, anisomycin, a competitive inhibitor of PMY binding to ribosomes (Pestka et al., 1972
; Sugita et al., 1995
; Hansen et al., 2003
), inhibits puromycylation ().
Properties of protein synthesis inhibitors
To further establish the biochemical mechanism of puromycylation in the presence of CHX or emetine, we developed an ELISA that measures puromycylation on nascent chains present on ribosomes extracted from cells. We isolated ribosomes by fractionating HeLa cell lysates on a 15–50% sucrose gradient (Stephens et al., 2008
), bound gradient fractions to polyvinylidene fluoride (PVDF) in a 96-well format, briefly incubated with PMY, and detected bound PMY by ELISA. This revealed that anti-PMY mAb binding is proportional to the amounts of polysomes (as opposed to monosomes) in the gradient fractions (). Concurrent ELISA for the large ribosome subunit acidic P proteins (consisting of P0, P1, and P2, for which highly specific antibodies in the form of human autoimmune anti–ribosomal P sera are commercially available) demonstrates that the failure to detect PMY in monosome (fraction 5) and free large subunit (fraction 4) fractions cannot be attributed to the lack of binding of ribosomes to PVDF. Treating cells with emetine before lysis increased the ratio of polysomes to monosomes and recapitulated the relationship between PMY binding and polysome abundance.
Anti-PMY immunoblotting of monosome- and polysome-containing fractions exposed to PMY in solution confirmed the lack of puromycylated proteins in the 80S (monosome) fraction and revealed parallel increases in puromycylated nascent chains with polysome sedimentation (). Blotting with anti–ribosomal P antibodies confirmed the presence of ribosomal proteins on the PVDF immunoblotting membrane for monosome fractions ().
These findings demonstrate that PMY binding to ribosomes correlates with the presence of puromycylated nascent chains and is not affected by emetine modification of ribosomes. Although PMY is capable of binding to ribosomes without puromycylation, we note that this interaction is of relatively low affinity (high micromolar) and is rapidly reversible (accounting for the reversibility of PMY on protein synthesis), which will not be detected when PMY is removed and ribosomes are subjected to multiple high volume washes. The low affinity binding of PMY with ribosomes is why previous investigators turned to N
-bromoacetyl PMY to permanently label ribosomes for structural experiments by covalently linking PMY to ribosomes (Lührmann et al., 1981
Functional evidence for stability of puromycylated nascent chain–ribosome association
Having established that PMY will only stably bind ribosomes via nascent chain puromycylation, we next used the surface sensing of translation (SUnSET) method (Schmidt et al., 2009
) to test whether puromycylated nascent chains are released in the presence of emetine. We pulsed cells for 10 min with PMY, washed to remove free PMY, incubated cells for 30 min at 37°C in the presence of various inhibitors, and measured the amount of puromycylated proteins transported to the cell surface via flow cytometry using 12D10 ( and S1 B; Schmidt et al., 2009
). Because puromycylated proteins reach the surface via the Golgi complex, blocking export from the ER with brefeldin A (BFA) sets the baseline level for complete blockade of nascent protein cell surface expression. As expected, 12D10 staining with cells incubated with BFA is equivalent to cells incubated at 4°C, which also prevents exocytosis.
Importantly, PMY labeling cells in the presence of emetine reduced PMY surface staining to background levels obtained with labeling with BFA, functionally demonstrating that emetine completely blocks release of puromycylated nascent chains by this measure. Adding emetine only during the chase provides a blockade indistinguishable from adding BFA only during the chase, showing that nascent chains remain associated with ribosomes for at least several minutes after puromycylation.
These findings demonstrate that emetine prevents the exocytosis of PMY-labeled nascent chains, consistent with the interpretation that they remain associated with ribosomes. Although SUnSET exclusively detects plasma membrane proteins with surface-exposed C termini, it is likely that other proteins are similarly retained on ribosomes, supporting the use of the RPM to detect translating ribosomes.
RPM visualizes active translation
We next visualized ribosome-associated nascent chains in cells via laser-scanning confocal microscopy by incubating HeLa cells with PMY and CHX for 5 min at 37°C (all the images that follow were generated by laser-scanning confocal microscopy). We then treated cells with digitonin to release PMY not associated with nascent chains, fixed cells with PFA, and performed standard indirect immunofluorescence with the anti-PMY mAb and anti–ribosomal P antibodies. Arsenite-induced translation inhibition reduced anti-PMY staining to background levels (). Emetine pretreatment increased staining, as opposed to its inhibition by anisomycin, which competes with PMY for binding to ribosomes.
Figure 2. Live-cell RPM detects translating ribosomes. (A) HeLa cells pretreated for 15 min with the inhibitor indicated were incubated with PMY (Puro) for 5 min at 37°C before digitonin extraction and PFA fixation. Samples were then incubated with 12D10 (more ...)
We correlated the effects of detergent extraction of ribosomes and puromycylated nascent chains on immunofluorescence versus immunoblotting (Fig. S2
). Digitonin treatment results in minimal loss of ribosomes and no detectable release of puromycylated nascent chains. In contrast, treating cells with NP-40 releases both ribosomes and puromycylated nascent chains and greatly reduces cytoplasmic immunofluorescence (see next section). This provides biochemical evidence supporting the conclusion that puromycylated nascent chains remain tethered to ribosomes.
High resolution confocal imaging () shows considerable but incomplete colocalization of PMY and ribosomal P, expected because of the absence of ribosomal P from some ribosomes (Gonzalo and Reboud, 2003
) as well as the detection of nontranslating monosomes, free ribosome heavy subunits, and free ribosomal P by anti–ribosomal P antibodies. The ability of the RPM to discriminate translating from nontranslating ribosomes is demonstrated in vaccinia virus (VV)–infected cells (). VV, like many viruses, shuts down host protein synthesis and shifts translation to viral mRNAs. VV assembles in cytoplasmic “factories,” identified by cytoplasmic DNA (stained by Hoechst 3358; , arrowheads) that recruit ribosomes and translation factors (Katsafanas and Moss, 2007
), implicating factories as sites of viral protein synthesis. 7 h after infection with VV expressing the karyophilic fusion protein influenza A virus (IAV) nucleoprotein (NP)-mCherry, translation principally localizes to a large DNA containing a viral factory (, magnification Z1). Notably, many ribosomes remain outside of viral factories and now do not stain with anti-PMY antibodies, indicating the specificity of the RPM for translating ribosomes. Anti-PMY immunoblotting of PMY-exposed VV-infected versus uninfected HeLa cells confirms that VV infection results in a diminished overall translation rate caused by host shutdown and also a distinct pattern of protein puromycylation, consistent with translation of viral proteins ().
The RPM detects robust nuclear translation in HeLa cells
We frequently noted a clear above background but less intense PMY and ribosomal P staining of the HeLa cell nucleoplasm using the digitonin RPM protocol. Although nuclei harbor large numbers of ribosomes, particularly in nucleoli in which ribosomes are assembled, digitonin poorly permeabilizes the nuclear membrane and nucleoplasm (Griffiths, 1993
), limiting antibody access to the nucleus and particularly the nucleolus, which is not stained by antibodies to ribosomal P proteins (). NP-40 permeabilization of nuclear membranes before fixation greatly increased nuclear RPM staining, particularly in nucleoli identified by diminished DNA staining and binding of antibodies specific for the nucleolar protein fibrillarin (). A potential artifact of nuclear translation is that the puromycylated nuclear proteins detected are synthesized by cytoplasmic ribosomes and transported to the nucleus during the labeling period. This artifact is unlikely to be a problem with the RPM because, as we show functionally () and biochemically (Fig. S2), puromycylated nascent chains remain tethered to ribosomes by translation elongation inhibitors and cannot traffic to nuclei. To be more certain, however, we determined that incubating cells with PMY for as short as 10 s suffices to clearly label the nuclear body and nucleoli, with intense staining occurring after only 30 s of PMY exposure ().
Figure 3. RPM detects nuclear/nucleolar translation. (A) HeLa cells were labeled live with emetine and PMY and processed for RPM staining using the regular digitonin extraction method or NP-40 extraction before fixation. (right) Higher magnification series of the (more ...)
Nuclear RPM is caused by nuclear protein synthesis and not import or trapping of cytoplasmic puromycylated nascent chains
Four lines of evidence support the conclusion that the RPM detects puromycylation of nascent chains in nuclei/nucleoli and not PMY bound to other cellular structures or puromycylated proteins that originate in the cytosol. First, CHX and emetine enhance RPM staining of the nucleoplasm and nucleolus (). If RPM nuclear staining is caused by transport of cytoplasmic puromycylated proteins, these inhibitors would reduce and not increase nuclear staining because they inhibit release of puromycylated proteins from cytoplasmic ribosomes. Second, anisomycin, which competes with PMY for A-site binding, blocks nuclear RPM staining, linking staining to ribosomal-based catalysis and essentially ruling out PMY association with other nuclear targets () because PMY and anisomycin are structurally dissimilar and unlikely to competitively bind the same off-target nuclear structure.
Third, harringtonine, which blocks initial peptide bond formation but allows completion of nascent chains and induces ribosome dissociation (), inhibits nuclear RPM staining, demonstrating that PMY staining does not reflect PMY binding to nuclear ribosomes (already extremely unlikely because of the low binding affinity of PMY for ribosomes in the absence of the nascent chain to puromycylate). Moreover, harringtonine inhibition of nuclear RPM staining (or cytoplasmic staining) is overridden if chain elongation is blocked by simultaneous addition of emetine (). Thus, harringtonine inhibition of nuclear RPM staining is caused by the specific absence of nascent chains on nuclear and cytoplasmic ribosomes and not other effects on cells. We made identical findings using emetine with arsenite as an alternative treatment to convert protein-synthesizing polysomes to translationally inactive monosomes ().
Fourth, blocking translation initiation via VV shutdown of host protein synthesis inhibits nuclear RPM staining in parallel with inhibiting synthesis outside of viral factories, when cells are examined 2, 4, and 5 h after infection (). Furthermore, nuclear RPM staining is blocked in parallel with the shutoff of host protein synthesis mediated by three RNA-different viruses. Vesicular stomatitis virus (VSV; a rhabdovirus; ) and Semliki forest virus (SFV; an alphavirus; ) nearly completely abrogate RPM staining of the nucleoplasm and nucleolus. IAV (an orthomyxovirus) selectively inhibits nucleolar RPM, sparing RPM staining in the nucleoplasm ().
Figure 4. Stress-regulated nuclear translation detected by RPM. (A) Uninfected HeLa cells or HeLa cells infected for 2, 4, or 5 h with rVV expressing NP-mCherry at a high MOI to infect all cells in fields before digitonin (cytoplasmic)- or NP-40 (nuclear)–based (more ...)
The four viruses examined inhibit host protein synthesis by distinct mechanisms (Bushell and Sarnow, 2002
), minimizing the possibility that their nuclear RPM staining inhibition is caused by other effects that the viruses have on host cell metabolism or structure. For each virus infection, nucleoli are intact, as clearly shown by either fibrillarin or ribosomal P staining, so the lack of nucleolar staining cannot be attributed to a lack of nucleoli themselves. For VV and SFV, which greatly reduce cytoplasmic RPM staining, surrounding uninfected cells provide an abundant source of puromycylated proteins yet do not result in nuclear staining of infected cells, providing further evidence against trapping of puromycylated cytoplasmic proteins in the nucleoplasm/nucleolus. The same is true for IAV and VSV, in which robust cytoplasmic RPM staining provides a more proximal source of puromycylated nascent chains.
To approximate the ratio of nuclear to cytoplasmic RPM staining, we developed a cofixation/NP-40 permeabilization modified protocol (simultaneously extracting cells with NP-40 while fixing with 3% PFA) that enables simultaneous visualization of staining in the nucleus and cytoplasm and recapitulates the enhancing effect of emetine and inhibiting effects of harringtonine and arsenite on a nuclear/cytoplasmic RPM (). Notably, RPM nuclear and cytoplasmic signals were similar to the values obtained via sequential procedures using NP-40 and digitonin, respectively. The cofixation/permeabilization method confirmed that nuclear RPM staining represents a significant fraction of a total cellular RPM.
Using this method, we also found a selective effect of IAV in blocking nucleolar but not nucleoplasmic or cytoplasmic RPM staining. This provides strong evidence against nucleolar trapping of puromycylated proteins translated elsewhere, particularly because IAV encodes seven proteins targeted to the nucleus (NS1, NP, M1, PB1-F2, and the three polymerases), three of which are translated at high levels (NS1, NP, and M1).
To “chase” nucleolar RPM staining, we incubated cells with anisomycin after PMY pulse. This was necessary to block further puromycylation, whose occurrence we hypothesize reflects the creation of an intracellular PMY depot during pulsing. After 6-h incubation, nucleolar PMY staining was significantly diminished, whereas staining in the nucleoplasm was constant (). Even in the absence of anisomycin during the chase, the balance of RPM staining moves from the nucleoli to the nuclear body (), providing additional evidence that the nucleolar RPM staining is not caused by import or trapping of puromycylated proteins from the nucleoplasm.
Figure 5. Nucleolar RPM labeling can be chased. (A) HeLa cells were pulse labeled for 5 min with PMY in the presence of CHX and then chased for 6 h in the presence of anisomycin to prevent further PMY incorporation. For time 0 and time 6 h, cells were RPM stained (more ...)
RPM staining detects nuclear translation in human monocytes
HeLa cells have been cultured for dozens of generations, are highly aneuploid, and are likely to demonstrate aberrant translation in some aspects relative to normal vertebrate cells. To show that nuclear translation is a normal process in human cells, we performed the RPM using purified blood monocytes from healthy donors (). As with HeLa cells, monocytes demonstrate clear staining of the nucleoplasm and nucleoli after 5-min exposure to PMY and emetine (). Moreover, anisomycin and harringtonine each inhibit both cytoplasmic and nuclear RPM staining (), recapitulating our previous observations in HeLa cells. Thus, nuclear translation also occurs in normal human cells ex vivo.
Figure 6. Nuclear RPM staining in monocytes. (A) Elutriated human peripheral blood monocytes pretreated or not pretreated with harringtonine 15 min before RPM staining (5-min PMY pulse in the presence of emetine) were fixed and extracted simultaneously with polysome (more ...)