Nematode translation is highly m7G cap-dependent
Three different luciferase reporters (Firefly, Renilla, and Guassia) were used to evaluate the contribution of the TMG cap and SL to RNA translation and stability in vivo. Reporter RNAs were synthesized in vitro with different caps (or no cap), 5’ UTRs, luciferase reporter open reading frames, 3’ UTRs, and poly(A) tails (or no tail) (see Suppl. Figure 1
for reporter characteristics). These RNAs were then biolistically introduced into ~800,000, 32–64 cell Ascaris embryos and luciferase activity assayed (Davis et al., 1999). The relative activities, time courses and dose responses were examined for all three reporters (; Suppl. Figure 2
; some data not shown). All three reporters demonstrated a linear dose response and were used at the lower end of the linear range (2.5 μg Firefly; 1 μg Renilla; 0.5 μg Gaussia). Maximal luciferase activity following introduction of the reporter RNA was typically reached at 60 min (Gaussia) or 90 min (Firefly and Renilla).
Luciferase RNA reporter dose response, activity over time, and comparison of reporter activities in Ascaris embryos
To provide a framework for the cap-dependence of nematode translation, we initially compared the translation of uncapped (pppG), GpppG-, and m7
GpppG-capped RNAs. Luciferase activity derived from uncapped (pppG) RNAs was not detectable whereas significant levels of luciferase activity were derived from m7
GpppG-capped RNAs (; Suppl. Figure 3
). Translation of GpppG-capped RNAs was also observed (Suppl. Figure 3
). However, GpppG-capped RNAs introduced into Ascaris cell-free extracts or embryos are rapidly N7-methylated [37
](data not shown). Addition of the methyl donor inhibitor SAH (S-adenosyl-homocysteine) prevents N7-methylation and eliminates translation of GpppG-capped RNA in the cell- free extracts. Overall, these data indicate that the GpppG-capped RNAs are not the direct substrates for translation.
Translation in Ascaris embryos is methyl-guanosine cap-dependent, enhanced by a poly(A) tail, and both a spliced leader and poly(A) tail are required for efficient translation of TMG-capped RNAs
In vivo methylation of GpppG-capped RNAs precluded our use of this cap as a baseline for guanine methylation-dependent translation. As an alternative, we examined ApppG-capped reporter RNAs, reasoning that the 5’ end of the mRNA would likely be more stable than pppG, as its 5’ –ppp- 5’ linkage would protect the 5’ end of the mRNA from degradation (). Translation of ApppG capped RNAs was detectable at a low level and therefore we used this capped RNA as a baseline for comparisons with other methylated, guanosine capped RNAs in subsequent experiments (–).
TMG cap does not support translation as efficiently as an m7GpppG cap
We next examined the individual contributions of N7- and N2-methylation of the guanosine cap to translation. Nematode embryo translation is highly m7
G-cap-dependent, as illustrated by the comparison between identical RNAs that differ only in an ApppG-vs. m7
GpppG-cap (–, ). Overall, translation of TMG-capped RNA (2 additional methyl groups at N2) was 2–3 fold lower than m7
G-capped RNA. The m7
GpppG-cap dependence and lower overall translation of TMG-capped RNA were observed with all three luciferase reporters and different 5’ and 3’ UTRs (see –; Suppl. Figure 3
; data not shown).
Effect of cap, poly(A) tail, and spliced leader sequence on translation of Renilla RNAs in Ascaris embryos
Contribution of m7GpppG vs m2,2,7GpppG cap, SL, and different 5’ and 3’ UTRs to translation in Ascaris embryos
Poly(A) tail synergizes with methylated cap to enhance translation
Addition of a 85 nt poly(A) tail greatly increased translation of either m7GpppG- or m32,2,7GpppG-capped RNAs, whereas the tail had only a small effect on ApppG-capped RNA ( and ). The effect of the methylated, guanosine cap and poly(A) tail together on translation are synergistic (). Interestingly, this synergistic effect is greater for RNAs that have both the TMG cap and SL (the trans-spliced elements) compared to m7GpppG-capped RNAs.
Ascaris embryo translation in vivo exhibits synergistic effects between the cap, poly(A) tail, and spliced leader sequence
SL facilitates translation of TMG-capped RNA
In order to determine the effect of the SL sequence itself on translation in vivo, we substituted the 5’ terminal 22 nt of the Renilla 5’ UTR with the SL sequence. The effect of this substitution was an ~50% decrease in overall translation of a m7GpppG-capped RNA ( and –). However, translation of m32,2,7GpppG-capped RNAs increased ~ 2-fold when the spliced leader was in the 5’ UTR. This 2-fold enhancement requires a poly(A) tail, as the same SL substitution in the 5’ UTR of a non-polyadenylated, m32,2,7GpppG-capped RNA had little effect on translation (Compare –, –). These data suggest the spliced leader has a positive, synergistic effect on the translation of TMG-capped RNAs, whereas when it is substituted into the 5’ UTR of m7GpppG-capped RNAs, the SL has a negative effect on translation (, –). Importantly, optimal translation of TMG-capped RNAs requires a downstream SL and a poly(A) tail. The data also indicate that the SL by itself does not act as a cap-independent enhancer of translation (–). Similar results were observed using Firefly reporter RNAs (data not shown).
Sequences between the SL and AUG in recipient mRNAs do not enhance translation of TMG-SL RNAs
The 22 nt nematode SL is typically added 0–10 nucleotides upstream of the AUG in the recipient RNA [37
]. To further examine the contribution of the SL and other downstream sequences between the SL and AUG in the 5’ UTR on translation, we evaluated native Ascaris 5’ UTRs of three trans-spliced mRNAs. RNA transfection efficiencies in these experiments were normalized by co-transfection of a control Firefly luciferase RNA. Consistent with the results in –, overall translation of TMG-capped reporter RNAs is considerably less compared to identical RNAs with an m7
G-cap unless the SL sequence is present at the 5’ end (–). Furthermore, sequences located between the SL and the initiation codon in native trans-spliced 5’ UTRs (SL-cDNA 12 and SL-cDNA 4) do not appear to play a large role in influencing translation of TMG-capped RNAs as illustrated by the comparison of the level of translation of the SL alone compared to SL-cDNA 12 or SL-cDNA 4 ().
To more closely examine the specificity of SL enhancement of the translation of TMG-capped RNAs, we compared the effect of the SL with non-trans-spliced 5’ UTRs (L37, Lg, and Sh)() and random sequences (N18-GAG and N21-ACC)() on the translation of TMG-capped RNA. Enhanced translation of TMG-capped RNA is observed only in the presence of the SL in the 5’ UTR. Overall, these data and those using three different luciferase reporter RNA 5’ UTRs (Firefly, Renilla, and Gaussia) demonstrate that the SL enhancement of TMG-capped RNA translation is a specific property of the SL sequence.
5’ UTR length and context affect translation
Overall translation for several reporter RNAs is also affected by the length of the 5’ UTR as illustrated by a comparison of the two Guassia 5’ UTRs (Sh vs Lg = Short vs Long)() and random sequence 5’ UTRs (data not shown). Reporters with shorter 5’ UTRs exhibited higher overall levels of translation compared to those with longer 5’ UTRs. The SL sequence immediately adjacent to an initiation codon (see SL in –) constitutes a relatively efficient 5’ UTR and initiation context for translation when directly compared to a Gaussia 5’ UTR of similar length (Sh). By comparison, the first 22 nts of the Firefly and Renilla 5’ UTRs provide a more efficient translation context in our experiments, as substitution of the SL for the first 22 nts of these 5’ UTRs led to a reduction in overall translation (– and ). Thus, a Gaussia 5’ UTR of the same length as the SL is only ~2/3 as efficient in translation when the two are directly compared. The lower level of translation of the Gaussia 5’ UTR is likely due in part to a combination of both its sequence and the -1 to -3 AUG context difference (SL = GAG, Gaussia = ACC). As shown in – (compare SL ACC, SL, GN21-ACC, and GN18-GAG), the SL GAG provides a more efficient -1 to -3 AUG context than ACC. In addition, the GAG context may contribute to the synergistic interaction between the SL and TMG cap. Thus, our comparisons of several different 5’ UTRs to the SL and its context demonstrate that, as observed in other systems, the length and sequence of the 5’ UTR contribute to translation efficiency in nematodes. However, in general the SL is not a significantly better component of the 5’ UTR than those sequences to which it was compared in the current study.
Native 3’ UTRs of trans-spliced mRNAs do not enhance translation of TMG-SL RNA
In experiments described above, the overall luciferase reporter translation in the context of the TMG cap and SL sequence was similar when different luciferase 3’ UTRs were present (Firefly, Renilla, and Gaussia 3’ UTRs). To determine whether the 3’ UTR of a trans-spliced mRNA contributes to the translation of TMG-SL mRNAs, we examined reporter RNAs with both the native 5’ and 3’ UTRs of two trans-spliced Ascaris mRNAs (cDNA 12, encoding vacuolar H+ ATPase 16 kd proteolipid subunit, and cDNA 4, encoding ribosomal protein L23)(). Reporter RNA activity was not significantly affected when both the native 5’ and 3’ UTR of these trans-spliced messages were present compared to only the trans-spliced 5’ UTR. Similar data were obtained with a Renilla and Firefly reporter (data not shown). These data demonstrate, that in the context of a luciferase reporter, the native 3’ UTRs did not cooperate with the SL or directly contribute to the in vivo translation of trans-spliced mRNAs.
TMG and SL do not play a differential role in 1–2 cell embryos
In the above studies, we examined the role of the TMG-cap and SL on translation in 32–64 cell embryos. To determine whether translation of trans-spliced mRNAs was the same in earlier embryos and/or in a discrete subset of embryo cells, we examined the translation of several mRNAs in 1–2 cell embryos. Translation of RNAs with TMG-cap alone compared to RNAs with an m7
GpppG-cap was even lower in early embryos. Overall, similar data were obtained for 1–2 cell embryos as observed for the 32–64 cell embryos described above (Supplementary Figure 4
TMG cap and SL together do not differentially affect RNA stability
The luciferase activity data presented above represent the sum of both RNA translation efficiency and stability. To differentiate the contributions of the TMG cap and SL on translation efficiency and functional RNA half-life, the kinetics of luciferase activity over time following RNA introduction into the embryos were determined (see Materials and Methods)[42
]. This methodology facilitates analysis of the independent contributions of RNA elements to translation and stability. This point is illustrated for 3 different poly(A) tail lengths in and Supplemental Figure 5
. Increasing poly(A) tail length acts to enhance translation and to increase functional RNA half-life. Thus, the increased overall level of translation as a function of poly(A) tail length is a function of both an increase in mRNA translation efficiency and functional RNA half-life.
Poly(A) tail affects both translation and stability in Ascaris embryos
As illustrated in previous experiments (), the SL or TMG cap added to a Renilla test RNA results in a reduction in translational efficiency (). The SL sequence substituted for sequences in the 5’ UTR of a Renilla test RNA causes a small decrease in mRNA stability. The reduction in both translation and stability by substitution of the SL by itself leads to an overall decrease in relative luciferase expression. Substitution of the m7G-cap with a TMG cap results in a significant decrease in translation efficiency, but does not affect functional mRNA half-life. Importantly, as shown in , the overall translational efficiencies and mRNA stabilities of non-trans-spliced (m7GpppG-Ren-A85) and trans-spliced test mRNAs (m2,2,7GpppG-SL-Ren-A85) are similar. Similar data were obtained with a Firefly reporter RNA and the functional half-lives of Gaussia reporter RNAs were not different (data not shown). In summary, the 1) luciferase activity data in previous sections are largely a function of overall translation efficiency and 2) RNAs that have both the TMG-cap and SL (representing trans-spliced RNA) do not have significantly different functional half-lives compared to similar RNAs with the m7GpppG cap (representing non-trans-spliced RNA).
Trans-spliced and non-trans-spliced mRNAs have similar translation efficiencies and functional half-lives following introduction into Ascaris embryos
To further compare the half-lives of these mRNAs, we also examined the physical stability of m7G-capped (non-trans-spliced) and TMG-capped SL (representing trans-spliced) reporter RNAs in Ascaris embryos using PCR (semi-quantitative PCR and real-time PCR of Renilla and Gaussia RNAs) and the decay of 32-P-labeled Gaussia transcripts. The decay slopes for the two RNAs were similar ( and data not shown). As shown in , levels of RNA drop dramatically within the first 10 minutes following RNA transfection. Following biolistic introduction of the RNAs, media added to the embryos contained RNase A to limit evaluation of RNA present in the media or adhering to the surface of the embryos from our analysis. We attribute the dramatic drop in RNA in the first 10 minutes to the decay or degradation of RNAs that are not incorporated into Ascaris embryo cells. Similar data were obtained using real-time PCR (data not shown). In addition, differences in the half-lives of RNAs determined using the kinetics of Firefly luciferase in and Gaussia physical half-life in are attributable to different luciferase RNAs (Renilla in and Gaussia in ) and the methods used to measure RNA half-lives. The important observation derived from these experiments is that trans-spliced and non-trans-spliced RNAs have similar half-lives in vivo demonstrating that the mRNA stabilities of trans-spliced and non-trans-spliced reporter RNAs are similar.
Physical half-life of trans-spliced vs non-trans-spliced test mRNA in Ascaris embryos
Real-time PCR analysis of luciferase RNAs over time following transfection enabled us to normalize luciferase activity to RNA levels in the embryos. These analyses (data not shown) led to similar data as those illustrated in – and and . Thus, overall translation differences observed were not a function of different levels of RNA introduced or present in the embryos.