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In Vitro Labeling Strategies for Single Ribonucleoprotein Machinery

 

Nature has long used large biomolecular assemblies to convert chemical energy into quasi-mechanical motion to perform essential functions of life. For example, cells use translation machinery -- the ribosome -- to make proteins from mRNA templates. Cellular nanomachines regulate the extent of translation by using miRNAs to guide AGO protein assembly into RISC to the 3' untranslated region. Similar to other machines, the ribonucleoprotein complex (RNP) requires the correct assembly of the correct number and type of molecules to perform its function. Any single misfolding or genetic mutation can lead to loss of function or abnormality and lead to disease. Intracellular single-molecule fluorescence microscopy (SMFM) imaging can be used to probe the structure and activity of individual machine components in their cellular environment. But the lack of high-yield methods to fluorescently label RNA molecules has hindered the development of SMFM.

 

Eukaryotic mRNAs are generally considered to be divided into four functional fragments: the 5' UTR (including the m 7 G cap), the 22, 23 coding sequences, the 3' UTR, and the poly(A) tail. Binding fluorescent probes to any of these fragments can be detrimental to the function of the mRNA, potentially leading to spurious findings and erroneous conclusions. Labeling strategies that can precisely bind a controllable number of fluorophores at the exact location of the RNA are therefore highly desirable. Chemical biologists from the University of Michigan used T7 RNA polymerase and yeast poly(A) polymerase to incorporate modified nucleotides into one of three segments of mRNA: randomly into the body (including the 5' UTR, coding region and 3 'UTR), randomly into the poly(A) tail, or specifically between the body and tail (BBT). After modification and labeling, each strategy was tested for its ability to generate mRNA using a combination of biochemical and single-molecule fluorescence microscopy methods.

 

T7 RNA Polymerase and Yeast Poly(A) Polymerase Efficiently Label mRNA Body and Tail, Respectively

To label firefly luciferase (FLuc) or Renilla luciferase (RLuc) mRNA, the scientists incorporated two chemically modified nucleoside triphosphates (CNTPs) into the RNA strand using an enzymatic labeling method: using T7 RNA polymerase Co-transcriptional incorporation of Cy5-UTP (Fig. 1A) and post-transcriptional incorporation of azido-ATP using the polymerase of yeast poly(A) (Fig. 1B). All three labeling strategies (Fig. 1C) were applied separately: 1) random incorporation of Cy5-UTP throughout the RNA [5' UTR, coding sequence and 3' UTR, (Fig. 1C top)]; 2) in bulk and poly (A) Continuous azido-ATP was added between the tails (middle of Figure 1C); 3) azido-ATP was randomly incorporated throughout the poly(A) tail (bottom of Figure 1C). Each strategy offers the unique ability to place fluorophores away from the region of interest.

Labeling of mRNAs

Figure 1. Labeling of mRNAs

 

They can tolerate CNTPs if the T7 RNA polymerase is altered at position 5. Therefore, supplementing the transcription reaction with Cy5-UTP will allow co-transcriptomic labeling of firefly luciferase mRNA [FLuc, (Figure 1C top)]. In addition, there was no increase in byproducts observed in 0% Cy5-UTP control transcripts (Figure 2A). Simultaneous quantification revealed a linear correlation between the number of fluorophores per purified RNA molecule and the fraction of Cy5-UTP during transcription (Figure 2B). This suggests that the extent of labeling can be easily predicted and controlled by adjusting the starting concentration of Cy5-UTP in the transcription mixture.

 

Labeling the RNA between its body and tail (BBT, Figure 1B) ensures that the CNTP is integrated outside of the functionally relevant sequence. For this strategy, RNA is first transcribed, 5' capped, and then the 3' position is modified with a continuous azido-ATP chain by yeast poly(A) polymerase (yPAP). After further polyadenylation (unmodified) and purification, RNA was labeled using Alexa647 reagent and the mild reaction conditions and high yield SPAAC click method. Gel analysis showed that the Alexa647 band intensity increased with time of incubation with yPAP and azide ATP (Figure 2C), resulting in a linear correlation especially at early time points (Figure 2D).

 

A third strategy is the random incorporation of azide-ATP during the poly(A) tailing step (Figure 1C bottom). This strategy is designed to save time and the number of necessary steps, which is critical when RNA yields are low. Gel analysis was performed by labeling the purified tail-modified RNA with Alexa647 with varying concentrations of azide-ATP (Figure 2E). The molar ratio of Alexa647 and RNA exhibited a linear correlation with the azido-ATP fraction in the ATP pool (Figure 2F). This strategy works best when tight control over the marking range is required. Additionally, to assess the effect of incorporated CNTPs on poly(A) tailing, body site and BBT-tagged mRNAs were assessed following yPAP-mediated tailing reactions. The results showed that regardless of the degree of labeling, body-labeled mRNAs were easily tailed, as shown by changes in molecular weight after yPAP treatment (Figure 2G). However, the azido-ATP modification in the BBT labeling strategy did not prevent yPAP from further modifying mRNAs with canonical poly(A) tails (Figure 2H).

Labeling range

Figure 2. Labeling range and post-transcriptional mRNA modifications for all three labeling strategies.

 

BBT and Tail Tags for mRNA Compatible with Translation

To test the compatibility of fluorescent mRNA with full-length translation, the investigators performed FLuc mRNA with each of three methods (Body = 60 labels/RNA, BBT = 17 labels/RNA, and Tail = 4.7 labels/RNA). Abundant labeling and incubation of the resulting mRNA in translation-capable extracts of rabbit reticulocytes. To detect all protein products, the reaction was supplemented with BIODIPY@-labeled lysine-bearing tRNA (Fluoro-TectTM), then quenched on gradient, 4–20% Tris-glycine SDS gels and analyzed for fluorescence of all molecular weights labeled proteins. Body-tagged mRNA produced 72% less FLuc protein compared to controls (Figure 3A), suggesting that this tagging strategy hinders ribosomal function. However, both BBT and tail labels produced protein bands with intensities within 10% of the control (Figure 3B). To assess their protein-coding capacity in cellulose, HeLa cells were transfected with heavily labeled BBT (17 labels/RNA) and tail (4.7 labels/RNA) modified FLuc mRNA, and their relative gene expression was measured ( Figure 3C). The results showed an approximately two-fold increase in FLuc expression of tagged mRNA compared to unmodified. Microinjection of labeled and unlabeled mRNA into U2OS and HeLa cells and detection of FLuc by immunofluorescence yielded the same results (Figure 4A,B). These data suggest that these two strategies are viable options for labeling mRNA and maintaining its encoded function.

BBT

Figure 3. BBT and tail-tagged mRNA produce translated protein.

Microinjected BBT

Figure 4. Microinjected BBT and tail-tagged mRNA selectively express FLuc protein.

 

3’ UTRs of BBT and Tail-Tagged mRNAs for miRNA Regulation

Since miRNAs are used to inhibit translation initiation, RISC-regulated mRNA targets loaded by miRNAs are characterized by repressed protein expression relative to their counterparts without corresponding target sites in their 3' UTRs. We designed a set of dual-luciferase reporter plasmids (RLuc and FLuc) that were engineered with 0, 1, 2, 3, 4, 5, 6, or 11 miR-7 miRNA recognition element (MRE) sites to Tagged mRNAs were tested for 3'UTR function. Transfection experiments with untagged mRNA showed that as the number of MREs increased in U2OS cells stably transfected with HeLa and DCP1a-EGFP, so did the repression of miRNA-dependent genes. Importantly, the tagged FLuc BBT (~17 tags/RNA) and tail (~5 tags/RNA) modified mRNAs with 0, 1, 3, 6 or 11 miR-7 MRE sites were transfected into HeLa cells showed the same increase in inhibitory signature (Fig. 5A). Therefore, the placement of these fluorophores can be compatible with RISC's miRNA-dependent translation initiation inhibition.

 

Another aspect of miRNA regulation of mRNA targets is their eventual degradation. mRNA destabilization involves recruitment of deadenylase complexes (CCR4-Not1 and PAN 2/3) for poly(A) tail shortening, DCP1/2 decapping complex, and XRN1-dependent 5'-to-3' mRNA digestion. These destabilizing processes are thought to occur in part in large cytoplasmic aggregates called P-Bodies. Therefore, recruitment of target mRNAs to P-Body particles is often regarded as a hallmark of degradation. We also created a U2OS cell line stably transfected and expressed DCP1a protein chimerically linked to EGFP and assessed the extent to which the microinjected labeled mRNA co-localized with EGFP-DCP1a 2 hours after injection (Figure 5B). After 2 h, live cells were imaged with Alexa647 foci within a 4-pixel radius of the P-Body centroid for more than 9 frames (0.9 s). At this threshold, approximately 50% of P-Bodies were found to co-localize with Alexa647-labeled mRNA in the presence of miR-7, regardless of labeling strategy (Fig. 5B,C). In contrast, in the absence of miR-7, an average of <10% of P-Bodies were found to co-localize with the mRNA (Fig. 5B,C). Taken together, using all three labeling strategies, the 3' UTR of mRNA can still be used for miRNA-dependent RNA silencing.

Fluorescent BBT

Figure 5. Fluorescent BBT and tail-tagged FLuc mRNA is inhibited and degraded by miRNA

 

 

Summarize

The researchers systematically tested and validated two enzymatic methods for the strategic in vitro integration of chemically modified nucleic acids (CNTPs) into one of three selected regions of the mRNA molecule: body, between body and tail (BBT). ) and the entire tail. Each strategy is unique and can place fluorophores in different regions of the RNA molecule depending on the needs of the experiment. They used two enzymes: T7 RNA polymerase and yeast poly(A) polymerase (yPAP). For body tagging, T7 RNA polymerase will contain a pyrimidine modified at position 5, such as Cy5-UTP. For BBT and tail modification methods, yPAP can efficiently incorporate small modifications such as azido-ATP at the 2' position of ATP. Fluorophore labeling of the azide moiety can be accomplished quickly and gently by incubating the modified RNA with the SPAAC fluorescent click reagent. Co-transcriptome (5' to 3' UTR) labeling of RNA molecules using T7 RNA polymerase is highly efficient and productive, but produces noncoding mRNA. To properly translate a viable protein, the highly processed components of the translation machinery must traverse the coding sequence unhindered. Two other strategies, BBT and tail tagging, were both efficient and productive, and were indistinguishable from their unmodified counterparts in protein-coding and miRNA-mediated regulation.