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Ribosomal proteins L5 and L15: Functional characterisation of important features, in vivo
Södertörn University, School of Life Sciences.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)Text
Abstract [en]

Protein synthesis is a highly regulated and energy consuming process, during which a large ribonucleoprotein particle called the ribosome, synthesizes new proteins. The eukaryotic ribosome consists of two unequal subunits called: small and large subunits. Both subunits are composed of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins).

Although rRNAs build the matrix of the ribosome and carries out catalysing of the peptide-bond formation between amino acids, r-proteins also appear to play important structural and functional roles. The primary role of r-proteins is to initiate the correct tertiary fold of rRNA and to organize the overall structure of the ribosome.

In this thesis, I focus on two proteins from the large subunit of the eukaryotic ribosome: r-proteins L5 and L15 from bakers yeast S. cerevisiae. Both r-proteins are essential for ribosome function. Their life cycle is primarily associated with rRNA interactions. As a consequence, the proteins show high sequence homology across the species borders. Furthermore, both L5 and L15 are connected to human diseases, which makes the study their role in ribosome biogenesis and ribosome function important.

By applying random- and site-directed mutagenesis, coupled with functional complementation tests, I aimed to elucidate functionally regions in both proteins, implicated in transport to the cell nucleus, protein-protein interactions and/or rRNA binding. The importance of individual and multiple amino acid exchanges in the primary sequence of rpL5 and rpL15 were studied in vivo. The obtained results show that S. cerevisiae rpL15 was tolerant to amino acid exchanges in the primary sequence, whereas rpL5 was not. Consequently, A. thaliana rpL15 could substitute for the function of wild type rpL15, whereas none of the tested orthologous proteins to rpL5 could substitute yeast rpL5 in vivo. These observations further emphasize the importance of studying r-proteins as separate entities in the ribosome context.

Place, publisher, year, edition, pages
Stockholm: Stockholm University , 2009. , 50 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:sh:diva-30701ISBN: 978-91-7155-896-1OAI: oai:DiVA.org:sh-30701DiVA: diva2:951038
Public defence
2009-06-16, MB 503, Södertörns högskola, Alfred Nobels allé 7, 10:00 (English)
Opponent
Supervisors
Available from: 2016-08-05 Created: 2016-08-05 Last updated: 2016-08-05Bibliographically approved
List of papers
1. Functional characterization of ribosomal protein L15 from Saccharomyces cerevisiae
Open this publication in new window or tab >>Functional characterization of ribosomal protein L15 from Saccharomyces cerevisiae
2009 (English)In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 55, no 2, 111-125 p.Article in journal (Refereed) Published
Abstract [en]

In this study we provide general information on the little studied eukaryotic ribosomal protein rpL15. Saccharomyces cerevisiae has two genes, YRPL15A and YRPL15B that could potentially code for yeast rpL15 (YrpL15). YRPL15A is essential while YRPL15B is dispensable. However, a plasmid-borne copy of the YRPL15B gene, controlled by the GAL1 promoter or by the promoter controlling expression of the YRPL15A gene, can functionally complement YrpL15A in yeast cells, while the same gene controlled by the authentic promoter is inactive. Analysis of the levels of YrpL15B-mRNA in yeast cells shows that the YRPL15B gene is inactive in transcription. The function of YrpL15A is highly resilient to single and multiple amino acid substitutions. In addition, minor deletions from both the N- and C-terminal ends of YrpL15A has no effect on protein function, while addition of a C-terminal tag that could be used for detection of plasmid-encoded YrpL15A is detrimental to protein function. YrpL15A could also be replaced by the homologous protein from Arabidopsis thaliana despite almost 30% differences in the amino acid sequence, while the more closely related protein from Schizosaccharomyces pombe was inactive. The lack of function was not caused by a failure of the protein to enter the yeast nucleus.

National Category
Genetics
Identifiers
urn:nbn:se:sh:diva-13904 (URN)10.1007/s00294-009-0228-z (DOI)000265092000002 ()2-s2.0-67349272028 (ScopusID)
Available from: 2011-12-14 Created: 2011-12-14 Last updated: 2016-08-05Bibliographically approved
2. Locating the nuclear localization signal in S. cerevisiae ribosomal protein L15A
Open this publication in new window or tab >>Locating the nuclear localization signal in S. cerevisiae ribosomal protein L15A
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Newly synthesized ribosomal proteins (r-proteins) are efficiently transported from the cytoplasm to the site of ribosome assembly in the nucleolus. Nuclear import is facilitated by the presence of a nuclear localization signal (NLS) whereas the nucleolar accumulation requires a nucleolar localization signal (NoLS). In this study we located the NLS of the essential r-protein YrpL15A by studying the ability of various YrpL15A-gfp constructs to enter the nucleus and nucleolus. We found that the NLS signal is located in the C-terminal part of the protein. The identified sequence was sufficient to direct the reporter construct to the nucleus in yeast cells. This protein fragment contains a sequence that resembles a classical monopartite NLS. The fragment also contains a NoLS as seen by the partial co-localization of reporter construct with the nucleolar marker protein nop1. Orthologs of YrpL15A such as rpL15B from Arabidopsis thaliana and rpL15A from Schizosaccharomyces pombe were also able to enter the nucleus and nucleolus of yeast cells, suggesting that their NLS and NoLS are similar to that found in YrpL15. These results are discussed in relation to sequence similarities/dissimilarities. YrpL15A containing a C-terminal tag was unable to assemble into large ribosomal subunits that were transported to the cytoplasm.

Keyword
Ribosomal protein L15, Functional complementation, Nuclear localization, Yeast
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-30699 (URN)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2009-05-14 Created: 2016-08-05 Last updated: 2016-08-05Bibliographically approved
3. Functional features of the C-terminal region of yeast ribosomal protein L5
Open this publication in new window or tab >>Functional features of the C-terminal region of yeast ribosomal protein L5
2008 (English)In: Molecular Genetics and Genomics, ISSN 1617-4615, E-ISSN 1617-4623, Vol. 280, no 4, 337-350 p.Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to analyze the functional importance of the C-terminus of the essential yeast ribosomal protein L5 (YrpL5). Previous studies have indicated that the C-terminal region of YrpL5 forms an alpha-helix with a positively charged surface that is involved in protein-5S rRNA interaction. Formation of an YrpL5 center dot 5S rRNA complex is a prerequisite for nuclear import of YrpL5. Here we have tested the importance of the alpha-helix and the positively charged surface for YrpL5 function in Saccharomyces cerevisiae using site directed mutagenesis in combination with functional complementation. Alterations in the sequence forming the putative alpha-helix affected the functional capacity of YrpL5. However, the effect did not correlate with a decreased ability of the protein to bind to 5S rRNA as all rpL5 mutants tested were imported to the nucleus whether or not the alpha-helix or the positively charged surface were intact. The alterations introduced in the C-terminal sequence affected the growth rate of cells expressing mutant but functional forms of YrpL5. The reduced growth rate was correlated with a reduced ribosomal content per cell indicating that the alterations introduced in the C-terminus interfered with ribosome assembly.

National Category
Biochemistry and Molecular Biology Genetics
Identifiers
urn:nbn:se:sh:diva-14121 (URN)10.1007/s00438-008-0369-7 (DOI)000258902300007 ()18751732 (PubMedID)2-s2.0-51349101530 (ScopusID)
Available from: 2011-12-18 Created: 2011-12-16 Last updated: 2016-08-05Bibliographically approved
4. Implications of N-terminal sequence elements in S. cerevisiae ribosomal protein L5
Open this publication in new window or tab >>Implications of N-terminal sequence elements in S. cerevisiae ribosomal protein L5
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Yeast ribosomal protein L5 (YrpL5) is an essential 5S rRNA-binding protein that forms the central protuberance of the large ribosomal subunit. Formation of the binary rpL5.5S rRNA complex is a prerequisite for nuclear import of rpL5 and for ribosome assembly. The involvment of the N-terminal sequences of YrpL5 in 5S rRNA interaction and nuclear import was studied by mutagenesis and functional complementation in S. cerevisiae. Furthermore, the ability of YrpL5 orthologous proteins from M. musculus (MrpL5), D. melanogaster (DrpL5) and A. thaliana (ArpL5) were non-functional in yeast cells. Nuclear import of YrpL5 requires conserved sequence elements in the N-terminus. Despite the presence of these elements in ArpL5, this protein was not recognized by the nuclear import machinery in yeast. This failure was probably due to lack of stable complex formation with yeast 5S rRNA.

Keyword
Functional complementation, Mutation analysis, Ribosomal protein L5, nuclear localization, S. cerevisiae
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-30700 (URN)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2009-05-14 Created: 2016-08-05 Last updated: 2016-08-05Bibliographically approved

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