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Systematic identification of non-coding RNA 2,2,7-trimethylguanosine cap structures in Caenorhabditis elegans.

Jia D, Cai L, He H, Skogerbø G, Li T, Aftab MN, Chen R - BMC Mol. Biol. (2007)

Bottom Line: All snRNAs known to have a TMG cap structure were found in the precipitate, indicating that our identification system was efficient.Further analysis showed that the SL RNAs and the Sm Y RNAs shared a very similar Sm binding site element (AAU4-5GGA), which sequence composition differed somewhat from those of other U snRNAs.Our results showed that most ncRNAs predicted to be transcribed by RNA polymerase II had a TMG cap, while those predicted to be transcribed by RNA plymerase III or located in introns did not have a TMG cap structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioinformatics Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. donging@gmail.com

ABSTRACT

Background: The 2,2,7-trimethylguanosine (TMG) cap structure is an important functional characteristic of ncRNAs with critical cellular roles, such as some snRNAs. Here we used immunoprecipitation with both K121 and R1131 anti-TMG antibodies to systematically identify the TMG cap structures for all presently characterized ncRNAs in C. elegans.

Results: The two anti-TMG antibodies precipitated a similar group of the C. elegans ncRNAs. All snRNAs known to have a TMG cap structure were found in the precipitate, indicating that our identification system was efficient. Other ncRNA families related to splicing, such as SL RNAs and Sm Y RNAs, were also found in the precipitate, as were 7 C/D box snoRNAs. Further analysis showed that the SL RNAs and the Sm Y RNAs shared a very similar Sm binding site element (AAU4-5GGA), which sequence composition differed somewhat from those of other U snRNAs. There were also 16 ncRNAs without an Sm binding site element in the precipitate, suggesting that for these ncRNAs, TMG formation may occur independently of Sm proteins.

Conclusion: Our results showed that most ncRNAs predicted to be transcribed by RNA polymerase II had a TMG cap, while those predicted to be transcribed by RNA plymerase III or located in introns did not have a TMG cap structure. Compared to ncRNAs without a TMG cap, TMG-capped ncRNAs tended to have higher expression levels. Five functionally non-annotated ncRNAs also have a TMG cap structure, which might be helpful for identifying the cellular roles of these ncRNAs.

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Precipitation efficiency of K121 and R1131 anti-TMG antibodies . (A) The number of ncRNAs that were assigned into TMG-capped group (TMG) or non-TMG-capped group (Non-TMG), or not assigned to either group (Other) by the K121 and R1131 anti-TMG antibodies. (B) Fluorescence intensity ratio distribution for the R1131 or K121 antibodies.
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Figure 1: Precipitation efficiency of K121 and R1131 anti-TMG antibodies . (A) The number of ncRNAs that were assigned into TMG-capped group (TMG) or non-TMG-capped group (Non-TMG), or not assigned to either group (Other) by the K121 and R1131 anti-TMG antibodies. (B) Fluorescence intensity ratio distribution for the R1131 or K121 antibodies.

Mentions: We first filtered and immunoprecipitated worm total RNA with the K121 anti-TMG antibody. However, after having been made aware that this antibody may have a reduced specificity for TMG caps and has shown some cross-reactivity with mono-methylated cap structures [25,26], we repeated all subsequent experiments with the R1131 antibody [17]. RNAs in both precipitate and supernatant obtained with both antibodies were extracted and reversely transcribed. The cDNA from precipitate and supernatant was labeled with Cy3 or Cy5, respectively, and hybridized to a microarray with probes for 127 ncRNAs [24]. For each of the 127 ncRNAs, the intensities of precipitation sample and supernatant sample were examined. Most of the ncRNAs had a clear tendency to occur either in the precipitation or in the supernatant sample. With a few exceptions, the two TMG antibodies precipitated the same ncRNAs, suggesting that both are able to effectively distinguish TMG capped ncRNAs from ncRNAs with other 5'-end structures (see below and Figure 1A). However, the data also indicated somewhat different specificities. For the snRNA U6, which have a simple γ-methylguanosine cap, a larger partition of this RNA was precipitated by K121 antibody (intensity ratio 0.55) than by R1131 antibody (intensity ratio 0.022; See Additional file 1). There was also a tendency towards precipitate and supernatant obtained with R1131 showing higher and lower intensity ratios, respectively, compared to those obtained with K121 (Figure 1B).


Systematic identification of non-coding RNA 2,2,7-trimethylguanosine cap structures in Caenorhabditis elegans.

Jia D, Cai L, He H, Skogerbø G, Li T, Aftab MN, Chen R - BMC Mol. Biol. (2007)

Precipitation efficiency of K121 and R1131 anti-TMG antibodies . (A) The number of ncRNAs that were assigned into TMG-capped group (TMG) or non-TMG-capped group (Non-TMG), or not assigned to either group (Other) by the K121 and R1131 anti-TMG antibodies. (B) Fluorescence intensity ratio distribution for the R1131 or K121 antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2200864&req=5

Figure 1: Precipitation efficiency of K121 and R1131 anti-TMG antibodies . (A) The number of ncRNAs that were assigned into TMG-capped group (TMG) or non-TMG-capped group (Non-TMG), or not assigned to either group (Other) by the K121 and R1131 anti-TMG antibodies. (B) Fluorescence intensity ratio distribution for the R1131 or K121 antibodies.
Mentions: We first filtered and immunoprecipitated worm total RNA with the K121 anti-TMG antibody. However, after having been made aware that this antibody may have a reduced specificity for TMG caps and has shown some cross-reactivity with mono-methylated cap structures [25,26], we repeated all subsequent experiments with the R1131 antibody [17]. RNAs in both precipitate and supernatant obtained with both antibodies were extracted and reversely transcribed. The cDNA from precipitate and supernatant was labeled with Cy3 or Cy5, respectively, and hybridized to a microarray with probes for 127 ncRNAs [24]. For each of the 127 ncRNAs, the intensities of precipitation sample and supernatant sample were examined. Most of the ncRNAs had a clear tendency to occur either in the precipitation or in the supernatant sample. With a few exceptions, the two TMG antibodies precipitated the same ncRNAs, suggesting that both are able to effectively distinguish TMG capped ncRNAs from ncRNAs with other 5'-end structures (see below and Figure 1A). However, the data also indicated somewhat different specificities. For the snRNA U6, which have a simple γ-methylguanosine cap, a larger partition of this RNA was precipitated by K121 antibody (intensity ratio 0.55) than by R1131 antibody (intensity ratio 0.022; See Additional file 1). There was also a tendency towards precipitate and supernatant obtained with R1131 showing higher and lower intensity ratios, respectively, compared to those obtained with K121 (Figure 1B).

Bottom Line: All snRNAs known to have a TMG cap structure were found in the precipitate, indicating that our identification system was efficient.Further analysis showed that the SL RNAs and the Sm Y RNAs shared a very similar Sm binding site element (AAU4-5GGA), which sequence composition differed somewhat from those of other U snRNAs.Our results showed that most ncRNAs predicted to be transcribed by RNA polymerase II had a TMG cap, while those predicted to be transcribed by RNA plymerase III or located in introns did not have a TMG cap structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioinformatics Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. donging@gmail.com

ABSTRACT

Background: The 2,2,7-trimethylguanosine (TMG) cap structure is an important functional characteristic of ncRNAs with critical cellular roles, such as some snRNAs. Here we used immunoprecipitation with both K121 and R1131 anti-TMG antibodies to systematically identify the TMG cap structures for all presently characterized ncRNAs in C. elegans.

Results: The two anti-TMG antibodies precipitated a similar group of the C. elegans ncRNAs. All snRNAs known to have a TMG cap structure were found in the precipitate, indicating that our identification system was efficient. Other ncRNA families related to splicing, such as SL RNAs and Sm Y RNAs, were also found in the precipitate, as were 7 C/D box snoRNAs. Further analysis showed that the SL RNAs and the Sm Y RNAs shared a very similar Sm binding site element (AAU4-5GGA), which sequence composition differed somewhat from those of other U snRNAs. There were also 16 ncRNAs without an Sm binding site element in the precipitate, suggesting that for these ncRNAs, TMG formation may occur independently of Sm proteins.

Conclusion: Our results showed that most ncRNAs predicted to be transcribed by RNA polymerase II had a TMG cap, while those predicted to be transcribed by RNA plymerase III or located in introns did not have a TMG cap structure. Compared to ncRNAs without a TMG cap, TMG-capped ncRNAs tended to have higher expression levels. Five functionally non-annotated ncRNAs also have a TMG cap structure, which might be helpful for identifying the cellular roles of these ncRNAs.

Show MeSH
Related in: MedlinePlus