Participation of Proteins of the CPSF complex in polyadenylation of transcripts read by RNA polymerase III from SINES

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

SINEs are mobile genetic elements of multicellular eukaryotes that arose during evolution from various tRNAs, as well as from 5S rRNA and 7SL RNA. Like the genes of these RNAs, SINEs are transcribed by RNA polymerase III. Transcripts of some mammalian SINEs have the ability to AAUAA-dependent polyadenylation that is unique for transcriptions generated by RNA polymerase III. Despite a certain similarity with canonical polyadenylation of mRNAs (transcripts of RNA polymerase II), these processes apparently differ significantly. The purpose of this work is to evaluate how important for polyadenylation of SINE transcripts are proteins of the CPSF complex formed by mPSF and mCF subcomplexes which directs mRNA polyadenylation. In HeLa cells, siRNA knockdowns of the CPSF components were carried out, after which the cells were transfected with plasmid constructs containing SINEs. A decrease of polyadenylation of the SINE transcripts as a result of the knockdown of the proteins was evaluated by Northern-hybridization. It turned out that the CPSF components, such as WDR33 and CPSF30, contributed to the polyadenylation of SINE transcriptions, while the knockdown of CPSF100, CPSF73 and symplekin did not reduce the polyadenylation of these transcripts. Wdr33 and CPSF30, along with the CPSF160 and Fip1 previously studied, are components of the subcomplex mPSF responsible for mRNA polyadenylation. Thus, the available data suggest the importance of all mPSF proteins for SINE transcriptions. At the same time, the CPSF100, CPSF73, and symplekin, forming the subcomplex mCF, are responsible for the cleavage of pre-mRNA, therefore, their non-participation in the polyadenylation of SINE transcriptions seems quite natural.

Full Text

Restricted Access

About the authors

I. G. Ustyantsev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: kramerov@eimb.ru
Russian Federation, Moscow, 119991

O. R. Borodulina

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kramerov@eimb.ru
Russian Federation, Moscow, 119991

D. A. Kramerov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: ustian@mail.ru
Russian Federation, Moscow, 119991

References

  1. Устьянцев И.Г., Голубчикова Ю.С., Бородулина О.Р., Крамеров Д.А. (2017) Каноническое и неканоническое полиаденилирование РНК. Молекуляр. биология. 51, 262–273. doi: 10.1134/S0026893317010186
  2. Sun Y., Hamilton K., Tong L. (2020) Recent molecular insights into canonical pre-mRNA 3’-end processing. Transcription. 11, 83–96.
  3. Liu J., Lu X., Zhang S., Yuan L., Sun Y. (2022) Molecular insights into mRNA polyadenylation and deadenylation. Int. J. Mol. Sci. 23, 10985.
  4. Boreikaite V., Passmore L.A. (2023) 3’-End processing of eukaryotic mRNA: machinery, regulation, and impact on gene expression. Annu. Rev. Biochem. 92, 199–225.
  5. Proudfoot N.J. (2011) Ending the message: poly(A) signals then and now. Genes Dev. 25, 1770–1782.
  6. Gutierrez P.A., Wei J., Sun Y., Tong L. (2022) Molecular basis for the recognition of the AUUAAA polyadenylation signal by mPSF. RNA. 28, 1534–1541.
  7. Zhang Y., Sun Y., Shi Y., Walz T., Tong L. (2020) Structural insights into the human pre-mRNA 3’-end processing machinery. Mol. Cell. 77, 800–809.e806.
  8. Yang Q., Gilmartin G.M., Doublie S. (2010) Structural basis of UGUA recognition by the Nudix protein CFI(m)25 and implications for a regulatory role in mRNA 3’ processing. Proc. Natl. Acad. Sci. USA. 107, 10062–10067.
  9. Zhu Y., Wang X., Forouzmand E., Jeong J., Qiao F., Sowd G.A., Engelman A.N., Xie X., Hertel K.J., Shi Y. (2018) Molecular mechanisms for CFIm-mediated regulation of mRNA alternative polyadenylation. Mol. Cell. 69, 62–74.e64.
  10. Sachs A., Wahle E. (1993) Poly(A) tail metabolism and function in eucaryotes. J. Biol. Chem. 268, 22955–22958.
  11. Nicholson A.L., Pasquinelli A.E. (2019) Tales of detailed poly(A) tails. Trends Cell Biol. 29, 191–200.
  12. Neve J., Patel R., Wang Z., Louey A., Furger A.M. (2017) Cleavage and polyadenylation: ending the message expands gene regulation. RNA Biol. 14, 865–890.
  13. Laishram R.S. (2014) Poly(A) polymerase (PAP) diversity in gene expression – star-PAP vs canonical PAP. FEBS Lett. 588, 2185–2197.
  14. Charlesworth A., Meijer H.A., de Moor C.H. (2013) Specificity factors in cytoplasmic polyadenylation. Wiley Interdiscip. Rev. RNA. 4, 437–461.
  15. Norbury C.J. (2013) Cytoplasmic RNA: a case of the tail wagging the dog. Nat. Rev. Mol. Cell Biol. 14, 643–653.
  16. Татосян К.А., Устьянцев И.Г., Крамеров Д.А. (2020) Деградация РНК в эукариотических клетках. Молекуляр. биология. 54, 542–561. doi: 10.1134/S0026893320040159
  17. Borodulina O.R., Kramerov D.A. (2008) Transcripts synthesized by RNA polymerase III can be polyadenylated in an AAUAAA-dependent manner. RNA. 14, 1865–1873.
  18. Kramerov D.A., Vassetzky N.S. (2011) SINEs. Wiley Interdiscip. Rev. RNA. 2, 772–786.
  19. Kramerov D.A., Vassetzky N.S. (2011) Origin and evolution of SINEs in eukaryotic genomes. Heredity. (Edinb). 107, 487–495.
  20. Vassetzky N.S., Kramerov D.A. (2013) SINEBase: a database and tool for SINE analysis. Nucl. Acids Res. 41, D83‒D89.
  21. Deininger P. (2011) Alu elements: know the SINEs. Genome Biol. 12, 236.
  22. Borodulina O.R., Kramerov D.A. (2001) Short interspersed elements (SINEs) from insectivores. Two classes of mammalian SINEs distinguished by A-rich tail structure. Mamm. Genome. 12, 779–786.
  23. Устьянцев И.Г., Татосян К.А., Стасенко Д.В., Кочанова Н.Ю., Бородулина О.Р., Крамеров Д.А. (2017) Полиаденилирование транскриптов, считываемых РНК-полимеразой III с SINE, значительно удлиняет время их жизни в клетке. Молекуляр. биология. 54, 78–86. doi: 10.1134/S0026893319040150
  24. Borodulina O.R., Ustyantsev I.G., Kramerov D.A. (2023) SINEs as potential expression cassettes: impact of deletions and insertions on polyadenylation and lifetime of B2 and Ves SINE transcripts generated by RNA polymerase III. Int. J. Mol. Sci. 24(19), 14600.
  25. Dewannieux M., Heidmann T. (2005) Role of poly(A) tail length in Alu retrotransposition. Genomics. 86, 378–381.
  26. Vassetzky N.S., Borodulina O.R., Ustyantsev I.G., Kosushkin S.A., Kramerov D.A. (2021) Analysis of SINE families B2, Dip, and Ves with special reference to polyadenylation signals and transcription terminators. Int. J. Mol. Sci. 22(18), 9897.
  27. Kosushkin S.A., Ustyantsev I.G., Borodulina O.R., Vassetzky N.S., Kramerov D.A. (2022) Tail wags dog’s SINE: retropositional mechanisms of can SINE depend on its a-tail structure. Biology (Basel). 11(10), 1403.
  28. Borodulina O.R., Golubchikova J.S., Ustyantsev I.G., Kramerov D.A. (2016) Polyadenylation of RNA transcribed from mammalian SINEs by RNA polymerase III: сomplex requirements for nucleotide sequences. Biochim. Biophys. Acta. 1859, 355–365.
  29. Ustyantsev I.G., Borodulina O.R., Kramerov D.A. (2021) Identification of nucleotide sequences and some proteins involved in polyadenylation of RNA transcribed by Pol III from SINEs. RNA Biol. 18, 1475–1488.
  30. Chomczynski P., Sacchi N. (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat. Protoc. 1, 581–585.
  31. Clerici M., Faini M., Muckenfuss L.M., Aebersold R., Jinek M. (2018) Structural basis of AAUAAA polyadenylation signal recognition by the human CPSF complex. Nat. Struct. Mol. Biol. 25, 135–138.
  32. Sun Y., Zhang Y., Hamilton K., Manley J.L., Shi Y., Walz T., Tong L. (2018) Molecular basis for the recognition of the human AAUAAA polyadenylation signal. Proc. Natl. Acad. Sci. USA. 115, E1419–E1428.
  33. Shi Y., Manley J.L. (2015) The end of the message: multiple protein-RNA interactions define the mRNA polyadenylation site. Genes Dev. 29, 889–897.
  34. Ray D., Kazan H., Cook K.B., Weirauch M.T., Najafabadi H.S., Li X., Gueroussov S., Albu M., Zheng H., Yang A., Na H., Irimia M., Matzat L.H., Dale R.K., Smith S.A., Yarosh C.A., Kelly S.M., Nabet B., Mecenas D., Li W., Laishram R.S., Qiao M., Lipshitz H.D., Piano F., Corbett A.H., Carstens R.P., Frey B.J., Anderson R.A., Lynch K.W., Penalva L.O., Lei E.P., Fraser A.G., Blencowe B.J., Morris Q.D., Hughes T.R. (2013) A compendium of RNA-binding motifs for decoding gene regulation. Nature. 499, 172–177.
  35. Mikula M., Bomsztyk K., Goryca K., Chojnowski K., Ostrowski J. (2013) Heterogeneous nuclear ribonucleoprotein (HnRNP) K genome-wide binding survey reveals its role in regulating 3’-end RNA processing and transcription termination at the early growth response 1 (EGR1) gene through XRN2 exonuclease. J. Biol. Chem. 288, 24788–24798.
  36. Wang Z., Qiu H., He J., Liu L., Xue W., Fox A., Tickner J., Xu J. (2020) The emerging roles of hnRNPK. J. Cell Physiol. 235, 1995–2008.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Additional materials to the article
Download (495KB)
Indexing metadata
3. Fig. 1. Decrease in protein expression levels following protein knockdown. a – Detection of proteins by Western blotting. siRNAs used to treat HeLa cells are indicated above the lanes. Names of the detected proteins are given below: left – control protein 40 kDa hnRNP E2, right – knockdown protein. b – Decrease in protein levels in cells treated with specific siRNAs relative to those in cells treated with control siRNA (corrected for expression of the control protein, hnRNP E2, in both samples). At least three knockdown experiments for each protein were performed, standard deviation is shown; * and ** – p < 0.05 and < 0.01, respectively (t-test).

Download (53KB)
Indexing metadata
4. Fig. 2. Effect of protein knockdown on polyadenylation of SINE transcripts. Northern blot analysis of RNA isolated from HeLa cells treated first with siRNA (control, anti-CPSF30, anti-Wdr33, anti-symplekin, anti-CPSF100, anti-CPSF73, anti-hnRNP K, anti-CFIm25, anti-CPSF160, or untreated siRNAs are indicated above the lanes) for 72 h and then transfected with different Ves, B2, or Ere SINE constructs (names are indicated below the lane series). Knockdown of CPSF160 or CFIm25 was performed as a positive control for decreased transcript polyadenylation. Black arrows indicate primary transcripts and curly brackets indicate polyadenylated transcripts; white arrow indicates a truncated Ves-specific transcript that results from transcription termination at a suboptimal terminator and is unable to polyadenylation. One of the lanes on the lower right blot – hnRNP K (48) – corresponds to an experiment in which transfection with plasmids was carried out 48 h after the start of treating the cells with siRNA to hnRNP K. The treatment time was shortened in order to reduce the cytotoxic effect of these siRNAs.

Download (76KB)
Indexing metadata
5. Fig. 3. Plots illustrating the quantitative effect of knockdown of different proteins on transcript polyadenylation of SINE B2, Ves, and Ere constructs containing only the τ signal (–Δβ), only the β signal (–Δτ), or both signals (–T, wild type). The average polyadenylation level of transcripts for each construct (names are indicated to the left of the plot) in cells transfected with specific siRNAs is shown relative to the polyadenylation level in cells treated with control siRNA (this level is taken as 100% and is marked with a vertical dotted line). The standard deviation is obtained from three experiments; *, **, and *** – p < 0.05, < 0.01, and < 0.001, respectively (t-test). a – Components of the mPSF subcomplex: CPSF30 and Wdr33. b – Components of the mCF subcomplex: symplekin, CPSF100 and CPSF73. c – hnRNP protein K.

Download (93KB)
Indexing metadata
6. Fig. 4. Scheme of polyadenylation of the SINE transcript synthesized by RNA polymerase III. Protein names are given in italics: proteins knocked down in our previous study [29] are shown in black, and proteins downregulated in this study are shown in red. The mPSF subcomplex of the CPSF complex interacts with the AAUAAA polyadenylation signal via the Wdr33 and CPSF30 subunits. Protein Y (CFIm25 in the case of SINE B2) binds to the τ signal, stimulating polyadenylation carried out by poly(A) polymerase (PAP). It is assumed that a hypothetical protein X, by binding to the β signal, also stimulates polyadenylation; in the case of B2, as well as Ves, this protein may be hnRNP K. The mCF subcomplex, responsible for cleavage of pre-mRNA, is not involved in the polyadenylation of the SINE transcript.

Download (27KB)
Indexing metadata

Copyright (c) 2024 Russian Academy of Sciences