Updating mRNA variants of the human RSK4 gene and their expression in different stressed situations

doi:10.1016/j.heliyon.2024.e27475

. 2024 Mar 8;10(7):e27475.

doi: 10.1016/j.heliyon.2024.e27475. eCollection 2024 Apr 15.

Updating mRNA variants of the human RSK4 gene and their expression in different stressed situations

Zhenwei Qin¹, Jianglin Yang^{2

3}, Keyin Zhang⁴, Xia Gao⁴, Qianchuan Ran¹, Yuanhong Xu¹, Zhi Wang⁴, Didong Lou¹, Chunhua Huang¹, Lucas Zellmer⁵, Guangxue Meng⁶, Na Chen⁶, Hong Ma⁶, Zhe Wang⁷, Dezhong Joshua Liao^{2

3}

Affiliations

¹ Section of Forensic Science and Pathology, School of Basic Medical Sciences, Guizhou University of Traditional Chinese Medicine, Dong-Qing-Nan Road, Guiyang, 550025, Guizhou Province, China.
² Center for Clinical Laboratories, The Affiliated Hospital of Guizhou Medical University, 4 Beijing Rd, Guiyang, 550004, Guizhou Province, China.
³ Key Lab of Endemic and Ethnic Diseases of the Ministry of Education of China in Guizhou Medical University, Guiyang, 550004, Guizhou Province, China.
⁴ Department of Pathology, The Affiliated Hospital of Guizhou Medical University, 4 Beijing Road, Guiyang, 550004, Guizhou Province, China.
⁵ Department of Medicine, Hennepin County Medical Center, 730 South 8th St., Minneapolis, MN, 55415, USA.
⁶ Department of Oral and Maxillofacial Surgery, School of Stomatology, Guizhou Medical University, 9 Beijing Road, Guiyang, 550004, Guizhou Province, China.
⁷ State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital, Air Force Medical University, 169 Changle West Road, Xi'an, 710032, China.

PMID: 38560189
PMCID: PMC10980951
DOI: 10.1016/j.heliyon.2024.e27475

Updating mRNA variants of the human RSK4 gene and their expression in different stressed situations

Zhenwei Qin et al. Heliyon. 2024.

. 2024 Mar 8;10(7):e27475.

doi: 10.1016/j.heliyon.2024.e27475. eCollection 2024 Apr 15.

Authors

Affiliations

¹ Section of Forensic Science and Pathology, School of Basic Medical Sciences, Guizhou University of Traditional Chinese Medicine, Dong-Qing-Nan Road, Guiyang, 550025, Guizhou Province, China.
² Center for Clinical Laboratories, The Affiliated Hospital of Guizhou Medical University, 4 Beijing Rd, Guiyang, 550004, Guizhou Province, China.
³ Key Lab of Endemic and Ethnic Diseases of the Ministry of Education of China in Guizhou Medical University, Guiyang, 550004, Guizhou Province, China.
⁴ Department of Pathology, The Affiliated Hospital of Guizhou Medical University, 4 Beijing Road, Guiyang, 550004, Guizhou Province, China.
⁵ Department of Medicine, Hennepin County Medical Center, 730 South 8th St., Minneapolis, MN, 55415, USA.
⁶ Department of Oral and Maxillofacial Surgery, School of Stomatology, Guizhou Medical University, 9 Beijing Road, Guiyang, 550004, Guizhou Province, China.
⁷ State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital, Air Force Medical University, 169 Changle West Road, Xi'an, 710032, China.

PMID: 38560189
PMCID: PMC10980951
DOI: 10.1016/j.heliyon.2024.e27475

Abstract

We determined RNA spectrum of the human RSK4 (hRSK4) gene (also called RPS6KA6) and identified 29 novel mRNA variants derived from alternative splicing, which, plus the NCBI-documented ones and the five we reported previously, totaled 50 hRSK4 RNAs that, by our bioinformatics analyses, encode 35 hRSK4 protein isoforms of 35-762 amino acids. Many of the mRNAs are bicistronic or tricistronic for hRSK4. The NCBI-normalized NM_014496.5 and the protein it encodes are designated herein as the Wt-1 mRNA and protein, respectively, whereas the NM_001330512.1 and the long protein it encodes are designated as the Wt-2 mRNA and protein, respectively. Many of the mRNA variants responded differently to different situations of stress, including serum starvation, a febrile temperature, treatment with ethanol or ethanol-extracted clove buds (an herbal medicine), whereas the same stressed situation often caused quite different alterations among different mRNA variants in different cell lines. Mosifloxacin, an antibiotics and also a functional inhibitor of hRSK4, could inhibit the expression of certain hRSK4 mRNA variants. The hRSK4 gene likely uses alternative splicing as a handy tool to adapt to different stressed situations, and the mRNA and protein multiplicities may partly explain the incongruous literature on its expression and comports.

Keywords: Alternative splicing; Polycistron; Protein isoform; RSK4.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

**Fig. 1**
Depiction of our primer design strategy for detecting the RNA variants with deletion or insertion of exon(s). For instance, to detect a variant lacking exon 3 (E3), a forward 20-mer (F1) is designed using the last 18 Nts of exon 2 as its long 5′ part and the first 2 Nts of exon 4 as its short 3′ part. RT-PCR using this skewed F1 and a reverse primer (R1) located on exon 5 should amplify only the variant without exon 3. Alternatively, a reverse 20-mer (R2) can be designed to have its first 17 Nts (reverse-complementarily) targeting the first 17 Nts of exon 4 and its last 3 Nts targeting the last 3 Nts of exon 2. RT-PCR using this R2 and a forward primer (F2) located on exon 1 should also amplify only the variant without exon 3. However, this strategy may need modifications, even when the annealing temperature has been raised to a level that decreases the PCR efficiency, because sometimes the last several Nts of a primer may have homologue(s) in one or more regions of the RNA. For instance, our 39R reverse primer (Table 1) designed for specifically amplifying the variant lacking the first 39 Nts (the shaded and italicized sequence) of the penultimate exon (in capital letters, with its flanking exons in lowercase letters) targets the “aagCGGTATACTGCTGAACAA” (underlined) sequence, but its first four nucleotides have several homologues (several underlined AAGC sequences) in the vicinity of the to-be-amplified region. Fortunately, this 4-Nt mis-annealing melts when the annealing temperature is raised to 60 °C.

**Fig. 2**
The NCBI-illustrated hRSK4 mRNA variants. A: An image from NCBI database illustrating the exon-intron relationships of the ten mRNA variants. Each short bar or box denotes an exon while each hyphen connecting 2 bars or boxes indicates an intron, with arrows pointing to the 5′-3′ direction. Note that NCBI puts the NM_014496.5 on the top to project its Wt status and that the ten mRNAs differ mainly at the sites of transcriptional initiation and at the first exon(s). B: Depiction of the relationship between the NM_014496.5 and the NM_001330512.1. Boxes represent exons with the number inside or above a box indicating the number of Nts the exon has, whereas the hyphens between two boxes represent introns. The first exon (with its 5′ part grey-shaded) of the NM_014496.5 has 381 Nts while its last 117 Nts (black-shaded) constitute the first exon of the NM_001330512.1. The 2nd and 3rd exons (black-shaded) of the NM_001330512.1 are derived from the first intron of the NM_014496.5. The last exon of the NM_014496.5 and NM_001330512.1 has 6053 Nts and 6057 Nts, respectively. C: An image from NCBI database showing an hRSK4 mRNA that is assembled by NCBI with all 26 exons of the hRSK4 gene (ENSG00000072133) collected by Ensembl. We sketch each exon's number of Nts in this assembled mRNA. Note that its first two exons (grey-shaded) do not exist in any of the ten NCBI-listed mRNAs while its 3rd exon (black-shaded) is the first one of the NM_014496.5.

**Fig. 3**
An illustration from “www.aceview.org” showing the a, b, c, d, e, and f groups of hRSK4 ESTs collected by NCBI. Note that group b was then annotated as the normalized mRNA (NM), which differs from the NM_014496.5 and NM_001330512.1 shown in Fig. 2B. Mention should be made that the original image at the NCBI website already has the minor overlapping of some numbers seen in the figure.

**Fig. 4**
The hRSK4 RNAs documented in Ensembl database. A table from Ensembl lists two mRNA variants (RPS6KA6-201 and RPS6KA6-204) and four noncoding RNAs of the hRSK4 gene (RPS6KA6). An illustration from Ensembl shows relationships among these six RNA sequences and exon-intron relationships within each RNA. However, the two mRNAs shown in the illustration seem to be an earlier version, since they differ in length from the corresponding mRNA in the table.

**Fig. 5**
The hRSK4 variants that have been reported by us previously [34] but have not yet been documented in NCBI or Ensembl. NCBI previously listed only one hRSK4 mRNA, which was updated stepwise from the NM_014496.2 until the NM_014496.4, all having 117 Nts in its first exon and 6057 Nts in its last exon and ending with a poly-A tail. We identified an mRNA variant (DEL141) that had its 141-Nt penultimate exon deleted and another variant (DEL39) that skipped only the first 39 Nts (the grey part) of the penultimate exon. A third variant we identified (IN96) had a 96-Nt exon inserted between the exons 1 and 2. A fourth variant (DEL112) we identified lacked the 112-Nt exon from the now NM_001330512.1. The 96-Nt, 112-Nt, and 110-Nt exons (black-shaded) are all derived from the intron 1 of the NM_014496.4. In addition, we identified a transcriptional initiation site that had a 5′ extension of 222 Nts from the first Nt of the 117-Nt exon 1 (underlined sequence) of the NM_014496.4 (exon 1 of the now NM_001330512.1). We rename this variant as 5EXT52 for its 5′ extension of 52 Nts from the first Nt of the 381-Nt exon 1 of the now NM_014496.5. The exons 1, 2, and 3 (shaded sequences) of this variant have 158 Nts, 24 Nts, and 157 Nts, respectively, while the introns 1 and 2 have 82 Nts and 12 Nts, respectively.

**Fig. 6**
The 5′ sequence of the NM_014496.5 (Wt-1) and NM_001330512.1 (Wt-2) as well as the upstream ORF for a short hRSK4 isoform in the mRNAs listed in NCBI or identified by us. A: The last 117 Nts (underlined) of exon 1 of Wt-1 constitute the 1st exon of Wt-2, while the last 81 (italicized) of these 117 Nts encode the first 27 AAs (with the ATG start codon shaded and boldfaced) of the Wt-1 protein (NP_055311.1). Translation of the Wt-2 mRNA incepting from this ATG stops at a TAG stop codon in exon 2, yielding a short upstream ORF (shaded sequence) encoding a 43-AA hRSK4. Translation of the NCBI-annotated hRSK4 protein (NP_00131744.1) encoded by the Wt-2 mRNA actually starts from an ATG (shaded, boldfaced and italicized) at exon 3, with the 27 AAs encoded by this exon (italic sequence) differing from the first 27 AAs of the Wt-1 protein. B: Six NCBI-listed variants as well as several mRNA variants we identified encompass one or two short upstream ORFs (sequences shown) encoding hRSK4 peptides (sequence shown), most of which (shaded) is identical to the N-terminus of the Wt-1 hRSK4.

**Fig. 7**
Illustration of the differences among the long hRSK4 protein isoforms encoded by NCBI- and Ensembl-listed mRNAs. Only the different part is shown, located at the N-terminus. Note that the first 27 AAs of the protein encoded by the NM_01496.5 and ENST00000262752.5 differ from those of the protein encoded by the NM_001330512.1 and ENST00000620340.4. Also note that some mRNAs encode the same protein. Dashed lines denote a lack of the sequence.

**Fig. 8**
RT-PCR detection of hRSK4 mRNAs in a panel of human cell lines with primer pairs covering different regions of the coding region. A: RT-PCR assay using the primer pair F35/R830 detects the Wt-1 and DEL82WT1 in some of the cell lines, while the primer pair F156/R830 detects the Wt-2 and/or the XM_011530917.3. RT-PCR using the F1675/R2493 amplifies a region shared by most (“Most”) known variants and also two novel variants (DEL321 and DEL516). B: RT-PCR assays using either F35/R830 or F156/R830 cannot detect hRSK4 expression in OMEC cells but can detect expected amplicons in the HGC-27 cells included as a positive control. However, RT-PCR assays involving a forward primer located downstream of F156, either V20F, F450 or F1675, result in anticipated amplicons, suggesting that OMEC cells express an hRSK4 variant (INTR1) that is transcriptionally initiated from an intron (like the XM_011530920.3) and lacks a 5′ region. C: RT-PCR assays using different primer pairs as indicated detect several new hRSK4 RNA variants, besides the DEL141 reported by us previously. “M” is the marker of molecular weights.

**Fig. 9**
Sketch of novel hRSK4 mRNA variants identified by us. Boxes represent exons while hyphens connecting boxes represent introns. The number inside a box indicates the number of Nts the exon has, whereas “Δ” indicates a sequence deletion. In the DEL150, DEL240, DEL288, DEL1137-39, DEL1462, DEL1534, and DEL1752, the grey portion of a box indicates the deleted part of the exon. In the IN38, IN132, IN148, and IN159, the shaded exon or shaded part of an exon is derived from an intron. In IN112WT2*, the inserted 112-Nt exon is derived from a 115-Nt region of the intron that, compared with the inserted 112-Nt sequence in PANC-1 cells, has not only three additional Nts (CTT or TCT) but also three mismatched nucleotides as mutations or polymorphisms (these differences are shown in the Supplementary Document).

**Fig. 10**
Detection of hRSK4 mRNAs in oral squamous cell carcinoma tissues (C) and their relatively normal surrounding tissues (N). A: RT-PCR results from four paired cancer and surrounding normal tissues. cDNA of the hHPRT1 gene was used as a reference for the loading control, which shows that the RNA quality and/or quantity differs slightly between the cancer tissue and its normal counterpart in the cases 1, 2, and 4 wherein the hRSK4 level in the cancerous tissue was greatly decreased. B: In an additional case with only little RNA available, a novel mRNA variant, the DEL420WT1, is detected besides the Wt-1 mRNA and the DEL82WT1 variant; the ratios between different variants differ greatly in the cancerous tissue, compared with the corresponding ratios in the surrounding normal tissue. M: molecular marker.

**Fig. 11**
Effects of serum starvation or treatment with moxifloxacin or trovafloxacin (Tro) on hRSK4 expression. A and B: RT-PCR assays of HGC-27 cells deprived of serum for three days (A) or A2870 cells derived of serum for four days (B) detect different hRSK4 variants with indicated primer pairs. Note that combining serum deprivation with a higher ambient temperature (39 °C) significantly induces the levels of both Wt-1 and Wt-2 in these two cell lines as detected by most primer pairs used, although the RT-PCR assays involving V20F or F1675 that amplifies most variants (**Most**) do not detect obvious differences among different treatments. C: RT-PCR assays reveal that moxifloxacin at a relatively lower concentration (10–30 μM) decreases the levels of the Wt-1 and Wt-2 in A2780 cells, but a higher ambient temperature (39 °C) can counteract this reduction. However, increase of moxifloxacin concentration to 70–100 μM starts to induce these two mRNAs, especially when the cells were cultured at 39 °C. D: RT-PCR assays reveal that trovafloxacin at a concentration of 5 μM can decrease the levels of the Wt-1 and Wt-2 in SKOV-3 cells. “Wt 2/XM917” means that the amplicon could be the Wt-2 and/or the XM_011530917.3. “Wt 2, 423, 424, 917” denotes that the amplicon could be any one(s) of the Wt-2, XM_017029423.2, XM_017029424.2, and XM_011530917.3. “?” denotes that the amplicon in the gels requires further determination.

**Fig. 12**
Long-lasting effects of our clove infusion on the survival and growth of PANC-1 and PANC-28 cells. A and B: PANC-1 (A) or PANC-28 (B) cells cultured at 37 °C or 39 °C were treated with 0.05–0.3% of the clove and determined for the live cells using a CCK8 assay with the solvent-treated controls set as 100% survival. C and D: PANC-1 (C) and PANC-28 (D) cells seeded at 1000 cells per well in a 6-well plate and cultured at 37 °C or 39 °C were treated with the indicated concentration of clove or with DMEM (as the untreated control) or ethanol (as the solvent control). After three days of treatment the cells were cultured in a new medium without the clove or ethanol for five more days, followed by a crystal blue staining of the live cells. Note that there are notably more live PANC-1 cells at 39 °C without treatment or treated with the ethanol, whereas the PANC-28 cells showed the opposite, when compared with their counterpart at 37 °C. However, in both cell lines there are many fewer cells treated with the higher concentration (0.2%) of clove at 39 °C than at 37 °C.

**Fig. 13**
Effects of our clove infusion on hRSK4 expression in PANC-1 and PANC-28 cells. PANC-1 (A) and PANC-28 (B) cells cultured at 37 °C or 39 °C were treated with 0.1% or 0.2% of the clove or with 0.2% of the solvent (95% ethanol) for three days and then evaluated for hRSK4 expression using RT-PCR assays with the indicated primer pairs. “M” denotes markers of molecular weights. ‘Wt 2/XM917″ means that the amplicon can be the Wt-2 and/or the XM_011530917.3, whereas “Wt 2, 423, 424, 917” means that the amplicon can be any one(s) of the Wt-2, XM_017029423.2, XM_017029424.2, and XM_011530917.3. “?” denotes that the amplicon requires further determination.

**Fig. 14**
Illustration of the differences between the RSK4 protein isoforms encoded by the newly-identified mRNA variants and the Wt-1 (NP_055311.1) or Wt-2 (NP_00131744.1). Only the sequence around the different region is shown. Dashed line or dashes denote deleted AAs, whereas the AAs different from those in a Wt are boldfaced and underlined. When a variant encodes two or more hRSK4 isoforms, a number is tagged after a variant's name to indicate that it has this number of AAs. Note that all short isoforms end with unmatched AAs and that some short isoforms appear in several mRNA variants.

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References

1. He Y., Yuan C., Chen L., Lei M., Zellmer L., Huang H., Liao D.J. Transcriptional-readthrough RNAs Reflect the phenomenon of "A gene contains gene(s)" or "Gene(s) within a gene" in the human genome, and thus are not chimeric RNAs. Genes. 2018;9 doi: 10.3390/genes9010040. -pii: E40. - DOI - PMC - PubMed
1. Jia Y., Chen L., Ma Y., Zhang J., Xu N., Liao D.J. To know how a gene works, We need to Redefine it first but then, more importantly, to Let the cell Itself Decide how to transcribe and process its RNAs. Int. J. Biol. Sci. 2015;11:1413–1423. doi: 10.7150/ijbs.13436. - DOI - PMC - PubMed
1. Liu X., Wang Y., Yang W., Guan Z., Yu W., Liao D.J. Protein multiplicity can lead to misconduct in western blotting and misinterpretation of immunohistochemical staining results, creating much conflicting data. Prog. Histochem. Cytochem. 2016;51:51–58. doi: 10.1016/j.proghi.2016.11.001. - DOI - PubMed
1. Pan Q., Shai O., Lee L.J., Frey B.J., Blencowe B.J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat. Genet. 2008;40:1413–1415. doi: 10.1038/ng.259. - DOI - PubMed
1. Top O., Milferstaedt S.W.L., Van G.N., Hoernstein S.N.W., Özdemir B., Decker E.L., Reski R. Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms. Commun. Biol. 2021;4:964. doi: 10.1038/s42003-021-02486-3. 10.1038/s42003-021-02486-3. - DOI - PMC - PubMed

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[1] He Y., Yuan C., Chen L., Lei M., Zellmer L., Huang H., Liao D.J. Transcriptional-readthrough RNAs Reflect the phenomenon of "A gene contains gene(s)" or "Gene(s) within a gene" in the human genome, and thus are not chimeric RNAs. Genes. 2018;9 doi: 10.3390/genes9010040. -pii: E40. - DOI - PMC - PubMed

[2] He Y., Yuan C., Chen L., Lei M., Zellmer L., Huang H., Liao D.J. Transcriptional-readthrough RNAs Reflect the phenomenon of "A gene contains gene(s)" or "Gene(s) within a gene" in the human genome, and thus are not chimeric RNAs. Genes. 2018;9 doi: 10.3390/genes9010040. -pii: E40. - DOI - PMC - PubMed

[3] Jia Y., Chen L., Ma Y., Zhang J., Xu N., Liao D.J. To know how a gene works, We need to Redefine it first but then, more importantly, to Let the cell Itself Decide how to transcribe and process its RNAs. Int. J. Biol. Sci. 2015;11:1413–1423. doi: 10.7150/ijbs.13436. - DOI - PMC - PubMed

[4] Jia Y., Chen L., Ma Y., Zhang J., Xu N., Liao D.J. To know how a gene works, We need to Redefine it first but then, more importantly, to Let the cell Itself Decide how to transcribe and process its RNAs. Int. J. Biol. Sci. 2015;11:1413–1423. doi: 10.7150/ijbs.13436. - DOI - PMC - PubMed

[5] Liu X., Wang Y., Yang W., Guan Z., Yu W., Liao D.J. Protein multiplicity can lead to misconduct in western blotting and misinterpretation of immunohistochemical staining results, creating much conflicting data. Prog. Histochem. Cytochem. 2016;51:51–58. doi: 10.1016/j.proghi.2016.11.001. - DOI - PubMed

[6] Liu X., Wang Y., Yang W., Guan Z., Yu W., Liao D.J. Protein multiplicity can lead to misconduct in western blotting and misinterpretation of immunohistochemical staining results, creating much conflicting data. Prog. Histochem. Cytochem. 2016;51:51–58. doi: 10.1016/j.proghi.2016.11.001. - DOI - PubMed

[7] Pan Q., Shai O., Lee L.J., Frey B.J., Blencowe B.J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat. Genet. 2008;40:1413–1415. doi: 10.1038/ng.259. - DOI - PubMed

[8] Pan Q., Shai O., Lee L.J., Frey B.J., Blencowe B.J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat. Genet. 2008;40:1413–1415. doi: 10.1038/ng.259. - DOI - PubMed

[9] Top O., Milferstaedt S.W.L., Van G.N., Hoernstein S.N.W., Özdemir B., Decker E.L., Reski R. Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms. Commun. Biol. 2021;4:964. doi: 10.1038/s42003-021-02486-3. 10.1038/s42003-021-02486-3. - DOI - PMC - PubMed

[10] Top O., Milferstaedt S.W.L., Van G.N., Hoernstein S.N.W., Özdemir B., Decker E.L., Reski R. Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms. Commun. Biol. 2021;4:964. doi: 10.1038/s42003-021-02486-3. 10.1038/s42003-021-02486-3. - DOI - PMC - PubMed

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Updating mRNA variants of the human RSK4 gene and their expression in different stressed situations

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Updating mRNA variants of the human RSK4 gene and their expression in different stressed situations

Authors

Affiliations

Abstract

Conflict of interest statement

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Abstract

Conflict of interest statement

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